Lyme disease vaccines

ABSTRACT

The present invention relates to novel vaccines for the prevention or attenuation of Lyme disease. The invention further relates to isolated nucleic acid molecules encoding antigenic polypeptides of  Borrelia burgdorferi . Antigenic polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention additionally relates to diagnostic methods for detecting  Borrelia  gene expression.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/830,230, filed Sep. 27, 2001, which is the national stage ofInternational Application No. PCT/US98/12718, filed Jun. 18, 1998, whichclaims benefit of U.S. Provisional Application No. 60/057,483, filedSep. 3, 1997, 60/053,344, filed Jul. 22, 1997, 60/053,377, filed Jul.22, 1997, and 60/050,359, filed Jun. 20, 1997. U.S. ProvisionalApplication No. 60/057,483 is incorporated by reference herein in itsentirety.

REFERENCE TO SEQUENCE LISTING ON COMPACT DISC

This application refers to a “Sequence Listing” listed below, which isprovided as an electronic document on two identical compact discs(CD-R), labeled “Copy 1” and “Copy 2.” These compact discs each containthe file “PB481D1.ST25.txt” (1,364,231 bytes, created on Nov. 23, 2004),which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel vaccines for the prevention orattenuation of Lyme disease. The invention further relates to isolatednucleic acid molecules encoding antigenic polypeptides of Borreliaburgdorferi. Antigenic polypeptides are also provided, as are vectors,host cells and recombinant methods for producing the same. The inventionadditionally relates to diagnostic methods for detecting Borrelia geneexpression.

BACKGROUND OF THE INVENTION

Lyme disease (Steere, A. C., Proc. Natl. Acad. Sci. USA 91: 2378-2383(1991)), or Lyme borreliosis, is presently the most common human diseasein the United States transmitted by an arthropod vector (Center forDisease Control, Morbid. Mortal. Weekly Rep. 46(23): 531-535 (1997)).Further, infection of house-hold pets, such as dogs, is a considerableproblem.

While initial symptoms often include a rash at the infection point, Lymedisease is a multisystemic disorder that may include arthritic,carditic, and neurological manifestations. While antibiotics arecurrently used to treat active cases of Lyme disease, B. burgdorferipersists even after prolonged antibiotic treatment. Further, B.burgdorferi can persist for years in a mammalian host in the presence ofan active immune response (Straubinger, R. et al., J. Clin. Microbiol.35: 111-116 (1997); Steere, A., N. Engl. J. Med. 321: 586-596 (1989)).

Lyme disease is caused by the related tick-borne spirochetes classifiedas Borrelia burgdorferi sensu lato (including B. burgdorferi sensustricto, B. afzelii, B. garinii). Although substantial progress has beenmade in the biochemical, ultrastructural, and genetic characterizationof the organism, the spirochetal factors responsible for infectivity,immune evasion and disease pathogenesis remain largely obscure.

A number of antigenic B. burgdorferi cell surface proteins have beenidentified. These include the outer membrane surface proteins (Osp)OspA, OspB, OspC and OspD. OspA and OspB are encoded by tightly linkedtandem genes which are transcribed as a single transcriptional unit(Brusca, J. et al., J. Bacteriol. 173: 8004-8008 (1991)). Themost-studied B. burgdorferi membrane protein is OspA, a lipoproteinantigen expressed by borreliae in resting ticks and the most abundantprotein expressed in vitro by most borrelial isolates (Barbour, A. G.,et al., Infection & Immunity 41: 795-804 (1983); Howe, T. R., et al.,Science 227: 645 (1985)).

A number of different types of Lyme disease vaccines have been shown toinduce immunological responses. Whole-cell B. burgdorferi vaccines, forexample, have been shown to induce both immunological responses andprotective immunity in several animal models (Reviewed in Wormser, G.,Clin. Infect. Dis. 21: 1267-1274 (1995)). Further, passive immunity hasbeen demonstrated in both humans and other animals using B. burgdorferispecific antisera.

While whole-cell Lyme disease vaccines confer protective immunity inanimal models, use of such vaccines presents the risk that responsiveantibodies will produce an autoimmune response (Reviewed in Wormser, G.,supra). This problem is at least partly the result of the production ofB. burgdorferi specific antibodies which cross-react with hepatocytesand both muscle and nerve cells. B. burgdorferi heat shock proteins andthe 41-kd flagellin subunit are believed to contain antigens whichelicit production of these cross-reactive antibodies.

Single protein subunit vaccines for Lyme disease have also been tested.The cell surface proteins of B. burgdorferi are potential candidates foruse in such vaccines and several have been shown to elicit protectiveimmune responses in mammals (Probert, W. et al., Vaccine 15: 15-19(1997); Fikrig, E. et al., Infect. Immun. 63: 1658-1662 (1995);Langerman S. et al., Nature 372: 552-556 (1994); Fikrig, E. et al., J.Immunol. 148: 2256-2260 (1992)). Experimental OspA vaccines, forexample, have demonstrated efficacy in several animal models (Fikrig,E., et al., Proc. Natl. Acad. Sci. USA 89: 5418-5421 (1992); Johnson, B.J., et al., Vaccine 13: 1086-1094 (1996); Fikrig, E., et al., Infect.Immun. 60: 657-661 (1992); Chang, Y. F., et al., Infection & Immunity63: 3543-3549 (1995)), and OspA vaccines for human use are underclinical evaluation (Keller, D., et al., J. Am. Med. Assoc. 271:1764-1768 (1994); Van Hoecke, C., et al., Vaccine 14: 1620-1626 (1996)).Passive immunity is also conferred by antisera containing antibodiesspecific for the full-length OspA protein. Further, vaccination withplasmid DNA encoding OspA has been demonstrated to elicit protectiveimmune responses in mice (Luke, C. et al., J. Infect. Dis. 175: 91-97(1997); Zhong, W. et al., Eur. J. Immunol. 26: 2749-2757 (1996)).

Recent immunofluorescence assay observations indicate that during tickengorgement the expression of OspA by borreliae diminishes (deSilva, A.M., et al., J. Exp. Med. 183: 271-275 (1996)) while expression of otherproteins, exemplified by OspC, increases (Schwan, T. G., et al., Proc.Natl. Acad. Sci. USA 92: 2909-2913 (1985)). By the time of transmissionto hosts, spirochetes in the tick salivary glands express little or noOspA. This down-modulation of OspA appears to explain the difficultiesin demonstrating immune responses to this antigen early in infectionfollowing tick bites (Kalish, R. A., et al., Infect. Immun. 63:2228-2235 (1995); Gern, L., et al., J. Infect. Dis. 167: 971-975 (1993);Schiable, U. E., et al., Immunol. Lett. 36: 219-226 (1993)) or followingchallenge with limiting doses of cultured borreliae (Schiable, U. E., etal., Immunol. Lett. 36: 219-226 (1993); Barthold, S. W. and Bockenstedt,L. K., Infect. Immun. 61: 4696-4702 (1993)).

Furthermore, OspA-specific antibodies are ineffective if administeredafter a borrelial challenge delivered by syringe (Schiable, U. E., etal., Proc. Natl. Acad. Sci. USA 87: 3768-3772 (1990)) or tick bite(deSilva, A. M., et al., J. Exp. Med. 183: 271-275 (1996)). To beefficacious, OspA vaccines must elicit protective levels of antibodywhich are maintained throughout periods of tick exposure in order toblock borrelia transmission from the arthropod vector.

Vaccines in current use against other pathogens include invivo-expressed antigens which could boost anamnestic responses uponinfection, potentiate the action of immune effector cells andcomplement, and inhibit key virulence mechanisms. OspC is both expressedduring infection (Montgomery, R. R., et al., J. Exp. Med. 183: 261-269(1996)) and a target for protective immunity (Gilmore, R. D., et al.,Infect. Immun. 64: 2234-2239 (1996); Probert, W. S. and LeFebvre, R. B.,Infect. Immun. 62: 1920-1926 (1994); Preac-Mursic, V., et al., Infection20: 342-349 (1992)), but mice immunized with this protein were onlyprotected against challenge with the homologous borrelial isolate(Probert, W. S., et al., J. Infect. Dis. 175: 400-405 (1997)).Identification of in vivo-expressed, and broadly protective, antigens ofB. burgdorferi has remained elusive.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding the B. burgdorferi peptides havingthe amino acid sequences shown in Table 1. Thus, one aspect of theinvention provides isolated nucleic acid molecules comprisingpolynucleotides having a nucleotide sequence selected from the groupconsisting of: (a) a nucleotide sequence encoding any of the amino acidsequences of the full-length polypeptides shown in Table 1; (b) anucleotide sequence encoding any of the amino acid sequences of thefull-length polypeptides shown in Table 1 but minus the N-terminalmethionine residue, if present; (c) a nucleotide sequence encoding anyof the amino acid sequences of the truncated polypeptides shown in Table1; and (d) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), or (c) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical, to any of the nucleotide sequences in (a), (b), (c), or(d) above, or a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide in (a), (b), (c), or (d)above. This polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.Additional nucleic acid embodiments of the invention relate to isolatednucleic acid molecules comprising polynucleotides which encode the aminoacid sequences of epitope-bearing portions of a B. burgdorferipolypeptide having an amino acid sequence in (a), (b), or (c) above.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using these vectors for theproduction of B. burgdorferi polypeptides or peptides by recombinanttechniques.

The invention further provides isolated B. burgdorferi polypeptideshaving an amino acid sequence selected from the group consisting of: (a)an amino acid sequence of any of the full-length polypeptides shown inTable 1; (b) an amino acid sequence of any of the full-lengthpolypeptides shown in Table 1 but minus the N-terminal methionineresidue, if present; (c) an amino acid sequence of any of the truncatedpolypeptides shown in Table 1; and (d) an amino acid sequence of anepitope-bearing portion of any one of the polypeptides of (a), (b), or(c).

The polypeptides of the present invention also include polypeptideshaving an amino acid sequence with at least 70% similarity, and morepreferably at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%similarity to those described in (a), (b), (c), or (d) above, as well aspolypeptides having an amino acid sequence at least 70% identical, morepreferably at least 75% identical, and still more preferably 80%, 85%,90%, 95%, 96%, 97%, 98%, or 99% identical to those above; as well asisolated nucleic acid molecules encoding such polypeptides.

The present invention further provides a vaccine, preferably amulti-component vaccine comprising one or more of the B. burgdorferipolypeptides shown in Table 1, or fragments thereof, together with apharmaceutically acceptable diluent, carrier, or excipient, wherein theB. burgdorferi polypeptide(s) are present in an amount effective toelicit an immune response to members of the Borrelia genus in an animal.The B. burgdorferi polypeptides of the present invention may further becombined with one or more immunogens of one or more other borrelial ornon-borrelial organisms to produce a multi-component vaccine intended toelicit an immunological response against members of the Borrelia genusand, optionally, one or more non-borrelial organisms.

The vaccines of the present invention can be administered in a DNA form,e.g., “naked” DNA, wherein the DNA encodes one or more borrelialpolypeptides and, optionally, one or more polypeptides of anon-borrelial organism. The DNA encoding one or more polypeptides may beconstructed such that these polypeptides are expressed fusion proteins.

The vaccines of the present invention may also be administered as acomponent of a genetically engineered organism. Thus, a geneticallyengineered organism which expresses one or more B. burgdorferipolypeptides may be administered to an animal. For example, such agenetically engineered organism may contain one or more B. burgdorferipolypeptides of the present invention intracellularly, on its cellsurface, or in its periplasmic space. Further, such a geneticallyengineered organism may secrete one or more B. burgdorferi polypeptides.

The vaccines of the present invention may be co-administered to ananimal with an immune system modulator (e.g., CD86 and GM-CSF).

The invention also provides a method of inducing an immunologicalresponse in an animal to one or more members of the Borrelia genus,e.g., B. burgdorferi sensu stricto, B. afzelii, and B. garinii,comprising administering to the animal a vaccine as described above.

The invention further provides a method of inducing a protective immuneresponse in an animal, sufficient to prevent or attenuate an infectionby members of the Borrelia genus, comprising administering to the animala composition comprising one or more of the polypeptides shown in Table1, or fragments thereof. Further, these polypeptides, or fragmentsthereof, may be conjugated to another immunogen and/or administered inadmixture with an adjuvant.

The invention further relates to antibodies elicited in an animal by theadministration of one or more B. burgdorferi polypeptides of the presentinvention.

The invention also provides diagnostic methods for detecting theexpression of genes of members of the Borrelia genus in an animal. Onesuch method involves assaying for the expression of a gene encodingBorrelia peptides in a sample from an animal. This expression may beassayed either directly (e.g., by assaying polypeptide levels usingantibodies elicited in response to amino acid sequences shown inTable 1) or indirectly (e.g., by assaying for antibodies havingspecificity for amino acid sequences shown in Table 1). An example ofsuch a method involves the use of the polymerase chain reaction (PCR) toamplify and detect Borrelia nucleic acid sequences.

The present invention also relates to nucleic acid probes having all orpart of a nucleotide sequence shown in Table 1 which are capable ofhybridizing under stringent conditions to Borrelia nucleic acids. Theinvention further relates to a method of detecting one or more Borrelianucleic acids in a biological sample obtained from an animal, said oneor more nucleic acids encoding Borrelia polypeptides, comprising:

-   a) contacting the sample with one or more of the above-described    nucleic acid probes, under conditions such that hybridization    occurs, and-   b) detecting hybridization of said one or more probes to the    Borrelia nucleic acid present in the biological sample.

DETAILED DESCRIPTION

The present invention relates to recombinant antigenic B. burgdorferipolypeptides and fragments thereof. The invention also relates tomethods for using these polypeptides to produce immunological responsesand to confer immunological protection to disease caused by members ofthe genus Borrelia. The invention further relates to nucleic acidsequences which encode antigenic B. burgdorferi polypeptides and tomethods for detecting Borrelia nucleic acids and polypeptides inbiological samples. The invention also relates to Borrelia specificantibodies and methods for detecting such antibodies produced in a hostanimal.

Definitions

The following definitions are provided to clarify the subject matterwhich the inventors consider to be the present invention.

As used herein, the phrase “pathogenic agent” means an agent whichcauses a disease state or affliction in an animal. Included within thisdefinition, for examples, are bacteria, protozoans, fungi, viruses andmetazoan parasites which either produce a disease state or render ananimal infected with such an organism susceptible to a disease state(e.g., a secondary infection). Further included are species and strainsof the genus Borrelia which produce disease states in animals.

As used herein, the term “organism” means any living biological system,including viruses, regardless of whether it is a pathogenic agent.

As used herein, the term “Borrelia” means any species or strain ofbacteria which is members of the genus Borrelia. Included within thisdefinition are Borrelia burgdorferi sensu lato (including B. burgdorferisensu stricto, B. afzelii, B. garinii), B. andersonii, B. anserina, B.japonica, B. coriaceae, and other members of the genus Borreliaregardless of whether they are known pathogenic agents.

As used herein, the phrase “one or more B. burgdorferi polypeptides ofthe present invention” means the amino acid sequence of one or more ofthe B. burgdorferi polypeptides disclosed in Table 1. These polypeptidesmay be expressed as fusion proteins wherein the B. burgdorferipolypeptides of the present invention are linked to additional aminoacid sequences which may be of borrelial or non-borrelial origin. Thisphrase further includes fragments of the B. burgdorferi polypeptides ofthe present invention.

As used herein, the phrase “full-length amino acid sequence” and“full-length polypeptide” refer to an amino acid sequence or polypeptideencoded by a full-length open reading frame (ORF). An ORF may be definedas a nucleotide sequence bounded by stop codons which encodes a putativepolypeptide. An ORF may also be defined as a nucleotide sequence withina stop codon bounded sequence which contains an initiation codon (e.g.,a methionine or valine codon) on the 5′ end and a stop codon on the 3′end.

As used herein, the phrase “truncated amino acid sequence” and“truncated polypeptide” refer to a sub-sequence of a full-length aminoacid sequence or polypeptide. Several criteria may also be used todefine the truncated amino acid sequence or polypeptide. For example, atruncated polypeptide may be defined as a mature polypeptide (e.g., apolypeptide which lacks a leader sequence). A truncated polypeptide mayalso be defined as an amino acid sequence which is a portion of a longersequence that has been selected for ease of expression in a heterologoussystem but retains regions which render the polypeptide useful for usein vaccines (e.g., antigenic regions which are expected to elicit aprotective immune response).

Additional definitions are provided throughout the specification.

Explanation of Table 1

Table 1 lists B. burgdorferi nucleotide and amino acid sequences of thepresent invention. The nomenclature used therein is as follows:

“nt” refers to nucleotide sequences;

-   -   “aa” refers to amino acid sequences;    -   “f” refers to full-length nucleotide or amino acid sequences;        and    -   “t” refers to truncated nucleotide or amino acid sequences.

Thus, for example, the designation “f101.aa” refers to the full-lengthamino acid sequence of B. burgdorferi polypeptide number 101. Further,“f101.nt” refers to the full-length nucleotide sequence encoding thefull-length amino acid sequence of B. burgdorferi polypeptide number101.

Explanation of Table 2

Table 2 lists accession numbers for the closest matching sequencesbetween the polypeptides of the present invention and those availablethrough GenBank and GeneSeq databases. These reference numbers are thedatabase entry numbers commonly used by those of skill in the art, whowill be familiar with their denominations. The descriptions of thenomenclature for GenBank are available from the National Center forBiotechnology Information. Column 1 lists the gene or ORF of the presentinvention. Column 2 lists the accession number of a “match” genesequence in GenBank or GeneSeq databases. Column 3 lists the descriptionof the “match” gene sequence. Columns 4 and 5 are the high score andsmallest sum probability, respectively, calculated by BLAST.Polypeptides of the present invention that do not share significantidentity/similarity with any polypeptide sequences of GenBank andGeneSeq are not represented in Table 2. Polypeptides of the presentinvention that share significant identity/similarity with more than oneof the polypeptides of GenBank and GeneSeq are represented more thanonce.

Explanation of Table 3.

The B. burgdorferi polypeptides of the present invention may include oneor more conservative amino acid substitutions from natural mutations orhuman manipulation as indicated in Table 3. Changes are preferably of aminor nature, such as conservative amino acid substitutions that do notsignificantly affect the folding or activity of the protein. Residuesfrom the following groups, as indicated in Table 3, may be substitutedfor one another: Aromatic, Hydrophobic, Polar, Basic, Acidic, and Small,

Explanation of Table 4

Table 4 lists residues comprising antigenic epitopes of antigenicepitope-bearing fragments present in each of the full length B.burgdorferi polypeptides described in Table 1 as predicted by theinventors using the algorithm of Jameson and Wolf, (1988) Comp. Appl.Biosci. 4: 181-186. The Jameson-Wolf antigenic analysis was performedusing the computer program PROTEAN (Version 3.11 for the PowerMacIntosh, DNASTAR, Inc., 1228 South Park Street Madison, Wis.). B.burgdorferi polypeptide shown in Table 1 may one or more antigenicepitopes comprising residues described in Table 4. It will beappreciated that depending on the analytical criteria used to predictantigenic determinants, the exact address of the determinant may varyslightly. The residues and locations shown described in Table 4correspond to the amino acid sequences for each full length genesequence shown in Table 1 and in the Sequence Listing. Polypeptides ofthe present invention that do not have antigenic epitopes recognized bythe Jameson-Wolf algorithm are not represented in Table 2.

Selection of Nucleic Acid Sequences Encoding Antigenic B. burgdorferiPolypeptides

The present invention provides a select number of ORFs from thosepresented in the fragments of the Borrelia burgdorferi genome which mayprove useful for the generation of a protective immune response. Thesequenced B. burgdorferi genomic DNA was obtained from a sub-culturedisolate of ATCC Deposit No. 35210. The sub-cultured isolate wasdeposited on Aug. 8, 1997 at the American Type Culture Collection, 12301Park Lawn Drive, Rockville, Md. 20852, and given accession number202012.

Some ORFs contained in the subset of fragments of the B. burgdorferigenome disclosed herein were derived through the use of a number ofscreening criteria detailed below. The ORFs are generally bounded at theamino terminus by a methionine residue and at the carboxy terminus by astop codon.

Many of the selected sequences do not consist of complete ORFs. Althougha polypeptide representing a complete ORF may be the closestapproximation of a protein native to an organism, it is not alwayspreferred to express a complete ORF in a heterologous system. It may bechallenging to express and purify a highly hydrophobic protein by commonlaboratory methods. Some of the polypeptide vaccine candidates describedherein have been modified slightly to simplify the production ofrecombinant protein. For example, nucleotide sequences which encodehighly hydrophobic domains, such as those found at the amino terminalsignal sequence, have been excluded from some constructs used for invitro expression of the polypeptides. Furthermore, any highlyhydrophobic amino acid sequences occurring at the carboxy terminus havealso been excluded from the recombinant expression constructs. Thus, inone embodiment, a polypeptide which represents a truncated or modifiedORF may be used as an antigen.

While numerous methods are known in the art for selecting potentiallyimmunogenic polypeptides, many of the ORFs disclosed herein wereselected on the basis of screening all theoretical Borrelia burgdorferiORFs for several aspects of potential immunogenicity. One set ofselection criteria are as follows:

1. Type I signal sequence: An amino terminal type I signal sequencegenerally directs a nascent protein across the plasma and outermembranes to the exterior of the bacterial cell. Experimental evidenceobtained from studies with Escherichia coli suggests that the typicaltype I signal sequence consists of the following biochemical andphysical attributes (Izard, J. W. and Kendall, D. A. Mol. Microbiol. 13:765-773 (1994)). The length of the type I signal sequence isapproximately 15 to 25 primarily hydrophobic amino acid residues with anet positive charge in the extreme amino terminus. In addition, thecentral region of the signal sequence adopts an alpha-helicalconformation in a hydrophobic environment. Finally, the regionsurrounding the actual site of cleavage is ideally six residues long,with small side-chain amino acids in the −1 and −3 positions.

2. Type IV signal sequence: The type IV signal sequence is an example ofthe several types of functional signal sequences which exist in additionto the type I signal sequence detailed above. Although functionallyrelated, the type IV signal sequence possesses a unique set ofbiochemical and physical attributes (Strom, M. S. and Lory, S., J.Bacteriol. 174: 7345-7351 (1992)). These are typically six to eightamino acids with a net basic charge followed by an additional sixteen tothirty primarily hydrophobic residues. The cleavage site of a type IVsignal sequence is typically after the initial six to eight amino acidsat the extreme amino terminus. In addition, type IV signal sequencesgenerally contain a phenylalanine residue at the +1 site relative to thecleavage site.

3. Lipoprotein: Studies of the cleavage sites of twenty-six bacteriallipoprotein precursors has allowed the definition of a consensus aminoacid sequence for lipoprotein cleavage. Nearly three-fourths of thebacterial lipoprotein precursors examined contained the sequenceL-(A,S)-(G,A)-C at positions −3 to +1, relative to the point of cleavage(Hayashi, S. and Wu, H. C., J. Bioenerg. Biomembr. 22: 451-471 (1990)).

4. LPXTG motif: It has been experimentally determined that most anchoredproteins found on the surface of gram-positive bacteria possess a highlyconserved carboxy terminal sequence. More than fifty such proteins fromorganisms such as S. pyogenes, S. mutans, B. burgdorferi, S. pneumoniae,and others, have been identified based on their extracellular locationand carboxy terminal amino acid sequence (Fischetti, V. A., ASM News 62:405-410 (1996)). The conserved region consists of six charged aminoacids at the extreme carboxy terminus coupled to 15-20 hydrophobic aminoacids presumed to function as a transmembrane domain. Immediatelyadjacent to the transmembrane domain is a six amino acid sequenceconserved in nearly all proteins examined. The amino acid sequence ofthis region is L-P-X-T-G-X, where X is any amino acid.

An algorithm for selecting antigenic and immunogenic Borreliaburgdorferi polypeptides including the foregoing criteria was developed.The algorithm is similar to that described in U.S. patent applicationSer. No. 08/781,986, filed Jan. 3, 1997, which is fully incorporated byreference herein. Use of the algorithm by the inventors to selectimmunologically useful Borrelia burgdorferi polypeptides resulted in theselection of a number of the disclosed ORFs. Polypeptides comprising thepolypeptides identified in this group may be produced by techniquesstandard in the art and as further described herein.

Nucleic Acid Molecules

The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding the B. burgdorferi polypeptideshaving the amino acid sequences shown in Table 1, which were determinedby sequencing the genome of B. burgdorferi deposited as ATCC deposit no.202012 and selected as putative immunogens.

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of DNA sequences determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Unless otherwise indicated, each “nucleotide sequence” set forth hereinis presented as a sequence of deoxyribonucleotides (abbreviated A, G, Cand T). However, by “nucleotide sequence” of a nucleic acid molecule orpolynucleotide is intended, for a DNA molecule or polynucleotide, asequence of deoxyribonucleotides, and for an RNA molecule orpolynucleotide, the corresponding sequence of ribonucleotides (A, G, Cand U), where each thymidine deoxyribonucleotide (T) in the specifieddeoxyribonucleotide sequence is replaced by the ribonucleotide uridine(U). For instance, reference to an RNA molecule having a sequence ofTable 1 set forth using deoxyribonucleotide abbreviations is intended toindicate an RNA molecule having a sequence in which eachdeoxyribonucleotide A, G or C of Table 1 has been replaced by thecorresponding ribonucleotide A, G or C, and each deoxyribonucleotide Thas been replaced by a ribonucleotide U.

Nucleic acid molecules of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced synthetically. The DNAmay be double-stranded or single-stranded. Single-stranded DNA or RNAmay be the coding strand, also known as the sense strand, or it may bethe non-coding strand, also referred to as the anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

In addition, isolated nucleic acid molecules of the invention includeDNA molecules which comprise a sequence substantially different fromthose described above but which, due to the degeneracy of the geneticcode, still encode a B. burgdorferi polypeptides and peptides of thepresent invention (e.g. polypeptides of Table 1). That is, all possibleDNA sequences that encode the B. burgdorferi polypeptides of the presentinvention. This includes the genetic code and species-specific codonpreferences known in the art. Thus, it would be routine for one skilledin the art to generate the degenerate variants described above, forinstance, to optimize codon expression for a particular host (e.g.,change codons in the bacteria mRNA to those preferred by a mammalian orother bacterial host such as E. coli).

The invention further provides isolated nucleic acid molecules havingthe nucleotide sequence shown in Table 1 or a nucleic acid moleculehaving a sequence complementary to one of the above sequences. Suchisolated molecules, particularly DNA molecules, are useful as probes forgene mapping and for identifying B. burgdorferi in a biological sample,for instance, by PCR, Southern blot, Northern blot, or other form ofhybridization analysis.

The present invention is further directed to nucleic acid moleculesencoding portions or fragments of the nucleotide sequences describedherein. Fragments include portions of the nucleotide sequences of Table1 at least 10 contiguous nucleotides in length selected from any twointegers, one of which representing a 5′ nucleotide position and asecond of which representing a 3′ nucleotide position, where the firstnucleotide for each nucleotide sequence in Table 1 is position 1. Thatis, every combination of a 5′ and 3′ nucleotide position that a fragmentat least 10 contiguous nucleotides in length could occupy is included inthe invention. “At least” means a fragment may be 10 contiguousnucleotide bases in length or any integer between 10 and the length ofan entire nucleotide sequence of Table 1 minus 1. Therefore, included inthe invention are contiguous fragments specified by any 5′ and 3′nucleotide base positions of a nucleotide sequences of Table 1 whereinthe contiguous fragment is any integer between 10 and the length of anentire nucleotide sequence minus 1.

Further, the invention includes polynucleotides comprising fragmentsspecified by size, in nucleotides, rather than by nucleotide positions.The invention includes any fragment size, in contiguous nucleotides,selected from integers between 10 and the length of an entire nucleotidesequence minus 1. Preferred sizes of contiguous nucleotide fragmentsinclude 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides.Other preferred sizes of contiguous nucleotide fragments, which may beuseful as diagnostic probes and primers, include fragments 50-300nucleotides in length which include, as discussed above, fragment sizesrepresenting each integer between 50-300. Larger fragments are alsouseful according to the present invention corresponding to most, if notall, of the nucleotide sequences shown in Table 1 or of the B.burgdorferi nucleotide sequences of the plasmid clones listed inTable 1. The preferred sizes are, of course, meant to exemplify notlimit the present invention as all size fragments, representing anyinteger between 10 and the length of an entire nucleotide sequence minus1, are included in the invention. Additional preferred nucleic acidfragments of the present invention include nucleic acid moleculesencoding epitope-bearing portions of B. burgdorferi polypeptidesidentified in Table 4.

The present invention also provides for the exclusion of any fragment,specified by 5′ and 3′ base positions or by size in nucleotide bases asdescribed above for any nucleotide sequence of Table 1 or the plasmidclones listed in Table 1. Any number of fragments of nucleotidesequences in Table 1 or the plasmid clones listed in Table 1, specifiedby 5′ and 3′ base positions or by size in nucleotides, as describedabove, may be excluded from the present invention.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding epitope-bearing portions of the B.burgdorferi polypeptides shown in Table 1. Such nucleic acid fragmentsof the present invention include, for example, nucleic acid moleculesencoding polypeptide fragments comprising from about the amino terminalresidue to about the carboxy terminal residue of each fragment shown inTable 4. The above referred to polypeptide fragments are antigenicregions of particular B. burgdorferi polypeptides shown in Table 1.Methods for determining other such epitope-bearing portions for theremaining polypeptides described in Table 1 are well known in the artand are described in detail below.

In another aspect, the invention provides isolated nucleic acidmolecules comprising polynucleotides which hybridize under stringenthybridization conditions to a portion of a polynucleotide in a nucleicacid molecule of the invention described above, for instance, a nucleicacid sequence shown in Table 1. By “stringent hybridization conditions”is intended overnight incubation at 42 C in a solution comprising: 50%formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate), 0.50 mM sodiumphosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20g/ml denatured, sheared salmon sperm DNA, followed by washing thefilters in 0.1×SSC at about 65 C.

By polynucleotides which hybridize to a “portion” of a polynucleotide isintended polynucleotides (either DNA or RNA) which hybridize to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

Of course, polynucleotides hybridizing to a larger portion of thereference polynucleotide, for instance, a portion 50-100 nt in length,or even to the entire length of the reference polynucleotide, are alsouseful as probes according to the present invention, as arepolynucleotides corresponding to most, if not all, of a nucleotidesequence as shown in Table 1. By a portion of a polynucleotide of “atleast 20 nt in length,” for example, is intended 20 or more contiguousnucleotides from the nucleotide sequence of the reference polynucleotide(e.g., a nucleotide sequences as shown in Table 1). As noted above, suchportions are useful diagnostically either as probes according toconventional DNA hybridization techniques or as primers foramplification of a target sequence by PCR, as described, for instance,in Molecular Cloning, A Laboratory Manual, 2nd. edition, Sambrook, J.,Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989), the entire disclosure of whichis hereby incorporated herein by reference.

Since nucleic acid sequences encoding the B. burgdorferi polypeptides ofthe present invention are provided in Table 1, generatingpolynucleotides which hybridize to portions of these sequences would beroutine to the skilled artisan. For example, the hybridizingpolynucleotides of the present invention could be generatedsynthetically according to known techniques.

As indicated, nucleic acid molecules of the present invention whichencode B. burgdorferi polypeptides of the present invention may include,but are not limited to those encoding the amino acid sequences of thepolypeptides by themselves; and additional coding sequences which codefor additional amino acids, such as those which provide additionalfunctionalities. Thus, the sequences encoding these polypeptides may befused to a marker sequence, such as a sequence encoding a peptide whichfacilitates purification of the fused polypeptide. In certain preferredembodiments of this aspect of the invention, the marker amino acidsequence is a hexa-histidine peptide, such as the tag provided in a pQEvector (Qiagen, Inc.), among others, many of which are commerciallyavailable. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenientpurification of the resulting fusion protein.

Thus, the present invention also includes genetic fusions wherein the B.burgdorferi nucleic acid sequences coding sequences provided in Table 1are linked to additional nucleic acid sequences to produce fusionproteins. These fusion proteins may include epitopes of borrelial ornon-borrelial origin designed to produce proteins having enhancedimmunogenicity. Further, the fusion proteins of the present inventionmay contain antigenic determinants known to provide helper T-cellstimulation, peptides encoding sites for post-translationalmodifications which enhance immunogenicity (e.g., acylation), peptideswhich facilitate purification (e.g., histidine “tag”), or amino acidsequences which target the fusion protein to a desired location (e.g., aheterologous leader sequence). For instance, hexa-histidine provides forconvenient purification of the fusion protein. See Gentz et al. (1989)Proc. Natl. Acad. Sci. 86: 821-24. The “HA” tag is another peptideuseful for purification which corresponds to an epitope derived from theinfluenza hemagglutinin protein. See Wilson et al. (1984) Cell 37: 767.As discussed below, other such fusion proteins include the B.burgdorferi polypeptides of the present invention fused to Fc at the N-or C-terminus.

Post-translational modification of the full-length B. burgdorferi OspAprotein expressed in E. coli is believed to increase the immunogenicityof this protein. Erdile, L. et al., Infect. Immun. 61: 81-90 (1993). B.burgdorferi OspA when expressed in E. coli, for example, ispost-translationally modified in at least two ways. First, a signalpeptide is cleaved; second, lipid moieties are attached. The presence ofthese lipid moieties is believed to confer enhanced immunogenicity andresults in the elicitation of a strong protective immunologicalresponse.

Variant and Mutant Polynucleotides

The present invention thus includes nucleic acid molecules and sequenceswhich encode fusion proteins comprising one or more B. burgdorferipolypeptides of the present invention fused to an amino acid sequencewhich allows for post-translational modification to enhanceimmunogenicity. This post-translational modification may occur either invitro or when the fusion protein is expressed in vivo in a host cell. Anexample of such a modification is the introduction of an amino acidsequence which results in the attachment of a lipid moiety. Such a lipidmoiety attachment site of OspA, which is lipidated upon expression in E.coli, has been identified. Bouchon, B. et al., Anal. Biochem. 246: 52-61(1997).

Thus, as indicated above, the present invention includes genetic fusionswherein a B. burgdorferi nucleic acid sequence provided in Table 1 islinked to a nucleotide sequence encoding another amino acid sequence.These other amino acid sequences may be of borrelial origin (e.g.,another sequence selected from Table 1) or non-borrelial origin. Anexample of such a fusion protein is reported in Fikrig, E. et al.,Science 250: 553-556 (1990) where an OspA-glutathione-5-transferasefusion protein was produced and shown to elicit protective immunityagainst Lyme disease in immune competent mice.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the B. burgdorferi polypeptides shown in Table 1.Variants may occur naturally, such as a natural allelic variant. By an“allelic variant” is intended one of several alternate forms of a geneoccupying a given locus on a chromosome of an organism. Genes II, Lewin,B., ed., John Wiley & Sons, New York (1985). Non-naturally occurringvariants may be produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions. The substitutions, deletions or additions mayinvolve one or more nucleotides. These variants may be altered in codingregions, non-coding regions, or both. Alterations in the coding regionsmay produce conservative or non-conservative amino acid substitutions,deletions or additions. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the B. burgdorferi polypeptides disclosedherein or portions thereof. Also especially preferred in this regard areconservative substitutions.

The present application is further directed to nucleic acid molecules atleast 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acidsequence shown in Table 1. The above nucleic acid sequences are includedirrespective of whether they encode a polypeptide having B. burgdorferiactivity. This is because even where a particular nucleic acid moleculedoes not encode a polypeptide having B. burgdorferi activity, one ofskill in the art would still know how to use the nucleic acid molecule,for instance, as a hybridization probe. Uses of the nucleic acidmolecules of the present invention that do not encode a polypeptidehaving B. burgdorferi activity include, inter alia, isolating an B.burgdorferi gene or allelic variants thereof from a DNA library, anddetecting B. burgdorferi mRNA expression samples, environmental samples,suspected of containing B. burgdorferi by Northern Blot analysis.

Embodiments of the invention include isolated nucleic acid moleculescomprising a polynucleotide having a nucleotide sequence at least 90%identical, and more preferably at least 95%, 96%, 97%, 98% or 99%identical to (a) a nucleotide sequence encoding any of the amino acidsequences of the full-length polypeptides shown in Table 1; (b) anucleotide sequence encoding any of the amino acid sequences of thefull-length polypeptides shown in Table 1 but minus the N-terminalmethionine residue, if present; (c) a nucleotide sequence encoding anyof the amino acid sequences of the truncated polypeptides shown in Table1; and (d) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), or (c) above.

Preferred, are nucleic acid molecules having sequences at least 90%,95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shownin Table 1, which do, in fact, encode a polypeptide having B.burgdorferi protein activity By “a polypeptide having B. burgdorferiactivity” is intended polypeptides exhibiting activity similar, but notnecessarily identical, to an activity of the B. burgdorferi protein ofthe invention, as measured in a particular biological assay suitable formeasuring activity of the specified protein.

Due to the degeneracy of the genetic code, one of ordinary skill in theart will immediately recognize that a large number of the nucleic acidmolecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequences shown in Table 1 will encode apolypeptide having B. burgdorferi protein activity. In fact, sincedegenerate variants of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving B. burgdorferi protein activity. This is because the skilledartisan is fully aware of amino acid substitutions that are either lesslikely or not likely to significantly effect protein function (e.g.,replacing one aliphatic amino acid with a second aliphatic amino acid),as further described below.

The biological activity or function of the polypeptides of the presentinvention are expected to be similar or identical to polypeptides fromother bacteria that share a high degree of structuralidentity/similarity. Tables 2 lists accession numbers and descriptionsfor the closest matching sequences of polypeptides available throughGenBank and Derwent databases. It is therefore expected that thebiological activity or function of the polypeptides of the presentinvention will be similar or identical to those polypeptides from otherbacterial genuses, species, or strains listed in Table 2.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the B.burgdorferi polypeptide. In other words, to obtain a polynucleotidehaving a nucleotide sequence at least 95% identical to a referencenucleotide sequence, up to 5% (5 of 100) of the nucleotides in thereference sequence may be deleted, inserted, or substituted with anothernucleotide. The query sequence may be an entire sequence shown in Table1, the ORF (open reading frame), or any fragment specified as describedherein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to anucleotide sequence of the presence invention can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. See Brutlag et al.(1990) Comp. App. Biosci. 6: 237-245. In a sequence alignment the queryand subject sequences are both DNA sequences. An RNA sequence can becompared by first converting U's to T's. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB alignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only nucleotidesoutside the 5′ and 3′ nucleotides of the subject sequence, as displayedby the FASTDB alignment, which are not matched/aligned with the querysequence, are calculated for the purposes of manually adjusting thepercent identity score.

For example, a 90 nucleotide subject sequence is aligned to a 100nucleotide query sequence to determine percent identity. The deletionsoccur at the 5′ end of the subject sequence and therefore, the FASTDBalignment does not show a matched/alignment of the first 10 nucleotidesat 5′ end. The 10 unpaired nucleotides represent 10% of the sequence(number of nucleotides at the 5′ and 3′ ends not matched/total number ofnucleotides in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90nucleotides were perfectly matched the final percent identity would be90%. In another example, a 90 nucleotide subject sequence is comparedwith a 100 nucleotide query sequence. This time the deletions areinternal deletions so that there are no nucleotides on the 5′ or 3′ ofthe subject sequence which are not matched/aligned with the query. Inthis case the percent identity calculated by FASTDB is not manuallycorrected. Once again, only nucleotides 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of B.burgdorferi polypeptides or fragments thereof by recombinant techniques.

Recombinant constructs may be introduced into host cells using wellknown techniques such as infection, transduction, transfection,transvection, electroporation and transformation. The vector may be, forexample, a phage, plasmid, viral or retroviral vector. Retroviralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally will occur only incomplementing host cells.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

Preferred are vectors comprising cis-acting control regions to thepolynucleotide of interest. Appropriate trans-acting factors may besupplied by the host, supplied by a complementing vector or supplied bythe vector itself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression, which may be inducible and/or cell type-specific.Particularly preferred among such vectors are those inducible byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives.

Expression vectors useful in the present invention include chromosomal-,episomal- and virus-derived vectors, e.g., vectors derived frombacterial plasmids, bacteriophage, yeast episomes, yeast chromosomalelements, viruses such as baculoviruses, papova viruses, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such ascosmids and phagemids.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating site at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A available fromStratagene; pET series of vectors available from Novagen; and ptrc99a,pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia. Amongpreferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. Other suitable vectors will be readily apparent to theskilled artisan.

Among known bacterial promoters suitable for use in the presentinvention include the E. coli lacI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR and PL promoters and the trppromoter. Suitable eukaryotic promoters include the CMV immediate earlypromoter, the HSV thymidine kinase promoter, the early and late SV40promoters, the promoters of retroviral LTRs, such as those of the Roussarcoma virus (RSV), and metallothionein promoters, such as the mousemetallothionein-I promoter.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

Transcription of DNA encoding the polypeptides of the present inventionby higher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually aboutfrom 10 to 300 bp that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at bp 100 to 270, the cytomegalovirus early promoter enhancer,the polyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

For secretion of the translated polypeptide into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. The signals may beendogenous to the polypeptide or they may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. SeeBennett, D. et al., J. Molec. Recogn. 8: 52-58 (1995) and Johanson, K.et al., J. Biol. Chem. 270 (16): 9459-9471 (1995).

The B. burgdorferi polypeptides can be recovered and purified fromrecombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography, lectin chromatography and high performance liquidchromatography (“HPLC”) is employed for purification. Polypeptides ofthe present invention include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic or eukaryotic host, including, forexample, bacterial, yeast, higher plant, insect and mammalian cells.

Polypeptides and Fragments

The invention further provides isolated polypeptides having the aminoacid sequences in Table 1, and peptides or polypeptides comprisingportions of the above polypeptides. The terms “peptide” and“oligopeptide” are considered synonymous (as is commonly recognized) andeach term can be used interchangeably as the context requires toindicate a chain of at least to amino acids coupled by peptidyllinkages. The word “polypeptide” is used herein for chains containingmore than ten amino acid residues. All oligopeptide and polypeptideformulas or sequences herein are written from left to right and in thedirection from amino terminus to carboxy terminus.

As discussed in detail below, immunization using B. burgdorferi sensustricto isolate B31 decorin-binding protein elicits the production ofantiserum which confers passive immunity against Borrelia species andstrains which express divergent forms of this protein. Cassatt, D. etal., Protection of Borrelia burgdorferi Infection by Antibodies toDecorin-binding Protein, in VACCINES97, Cold Spring Harbor Press (1997),pages 191-195. Thus, some amino acid sequences of the B. burgdorferipolypeptides shown in Table 1 can be varied without significantlyeffecting the antigenicity of the polypeptides. If such differences insequence are contemplated, it should be remembered that there will becritical areas on the polypeptide which determine antigenicity. Ingeneral, it is possible to replace residues which do not form part of anantigenic epitope without significantly effecting the antigenicity of apolypeptide.

Variant and Mutant Polypeptides

To improve or alter the characteristics of B. burgdorferi polypeptidesof the present invention, protein engineering may be employed.Recombinant DNA technology known to those skilled in the art can be usedto create novel mutant proteins or muteins including single or multipleamino acid substitutions, deletions, additions, or fusion proteins. Suchmodified polypeptides can show, e.g., enhanced activity or increasedstability. In addition, they may be purified in higher yields and showbetter solubility than the corresponding natural polypeptide, at leastunder certain purification and storage conditions.

N-Terminal and C-Terminal Deletion Mutants

It is known in the art that one or more amino acids may be deleted fromthe N-terminus or C-terminus without substantial loss of biologicalfunction. For instance, Ron et al. J. Biol. Chem., 268: 2984-2988(1993), reported modified KGF proteins that had heparin binding activityeven if 3, 8, or 27 N-terminal amino acid residues were missing.Accordingly, the present invention provides polypeptides having one ormore residues deleted from the amino terminus of the amino acid sequenceof the B. burgdorferi polypeptides shown in Table 1, and polynucleotidesencoding such polypeptides.

Similarly, many examples of biologically functional C-terminal deletionmuteins are known. For instance, Interferon gamma shows up to ten timeshigher activities by deleting 8-10 amino acid residues from the carboxyterminus of the protein See, e.g., Dobeli, et al. (1988) J.Biotechnology 7: 199-216. Accordingly, the present invention providespolypeptides having one or more residues from the carboxy terminus ofthe amino acid sequence of the B. burgdorferi polypeptides shown inTable 1. The invention also provides polypeptides having one or moreamino acids deleted from both the amino and the carboxyl termini asdescribed below.

The present invention is further directed to polynucleotide encodingportions or fragments of the amino acid sequences described herein aswell as to portions or fragments of the isolated amino acid sequencesdescribed herein. Fragments include portions of the amino acid sequencesof Table 1, are at least 5 contiguous amino acid in length, are selectedfrom any two integers, one of which representing a N-terminal position.The initiation codon of the polypeptides of the present inventionsposition 1. Every combination of a N-terminal and C-terminal positionthat a fragment at least 5 contiguous amino acid residues in lengthcould occupy, on any given amino acid sequence of Table 1 is included inthe invention. At least means a fragment may be 5 contiguous amino acidresidues in length or any integer between 5 and the number of residuesin a full length amino acid sequence minus 1. Therefore, included in theinvention are contiguous fragments specified by any N-terminal andC-terminal positions of amino acid sequence set forth in Table 1 whereinthe contiguous fragment is any integer between 5 and the number ofresidues in a full length sequence minus 1.

Further, the invention includes polypeptides comprising fragmentsspecified by size, in amino acid residues, rather than by N-terminal andC-terminal positions. The invention includes any fragment size, incontiguous amino acid residues, selected from integers between 5 and thenumber of residues in a full length sequence minus 1. Preferred sizes ofcontiguous polypeptide fragments include about 5 amino acid residues,about 10 amino acid residues, about 20 amino acid residues, about 30amino acid residues, about 40 amino acid residues, about 50 amino acidresidues, about 100 amino acid residues, about 200 amino acid residues,about 300 amino acid residues, and about 400 amino acid residues. Thepreferred sizes are, of course, meant to exemplify, not limit, thepresent invention as all size fragments representing any integer between5 and the number of residues in a full length sequence minus 1 areincluded in the invention. The present invention also provides for theexclusion of any fragments specified by N-terminal and C-terminalpositions or by size in amino acid residues as described above. Anynumber of fragments specified by N-terminal and C-terminal positions orby size in amino acid residues as described above may be excluded.

The above fragments need not be active since they would be useful, forexample, in immunoassays, in epitope mapping, epitope tagging, togenerate antibodies to a particular portion of the protein, as vaccines,and as molecular weight markers.

Other Mutants

In addition to N- and C-terminal deletion forms of the protein discussedabove, it also will be recognized by one of ordinary skill in the artthat some amino acid sequences of the B. burgdorferi polypeptide can bevaried without significant effect of the structure or function of theprotein. If such differences in sequence are contemplated, it should beremembered that there will be critical areas on the protein whichdetermine activity.

Thus, the invention further includes variations of the B. burgdorferipolypeptides which show substantial B. burgdorferi polypeptide activityor which include regions of B. burgdorferi protein such as the proteinportions discussed below. Such mutants include deletions, insertions,inversions, repeats, and type substitutions selected according togeneral rules known in the art so as to have little effect on activity.For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided. There are two main approaches forstudying the tolerance of an amino acid sequence to change. See, Bowie,J. U. et al. (1990), Science 247: 1306-1310. The first method relies onthe process of evolution, in which mutations are either accepted orrejected by natural selection. The second approach uses geneticengineering to introduce amino acid changes at specific positions of acloned gene and selections or screens to identify sequences thatmaintain functionality.

These studies have revealed that proteins are surprisingly tolerant ofamino acid substitutions. The studies indicate which amino acid changesare likely to be permissive at a certain position of the protein. Forexample, most buried amino acid residues require nonpolar side chains,whereas few features of surface side chains are generally conserved.Other such phenotypically silent substitutions are described by Bowie etal. (supra) and the references cited therein. Typically seen asconservative substitutions are the replacements, one for another, amongthe aliphatic amino acids Ala, Val, Leu and Ile; interchange of thehydroxyl residues Ser and Thr, exchange of the acidic residues Asp andGlu, substitution between the amide residues Asn and Gln, exchange ofthe basic residues Lys and Arg and replacements among the aromaticresidues Phe, Tyr.

Thus, the fragment, derivative, analog, or homolog of the polypeptide ofTable 1, or that encoded by the plasmids listed in Table 1, may be: (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code: or (ii) one in which one or moreof the amino acid residues includes a substituent group: or (iii) one inwhich the B. burgdorferi polypeptide is fused with another compound,such as a compound to increase the half-life of the polypeptide (forexample, polyethylene glycol): or (iv) one in which the additional aminoacids are fused to the above form of the polypeptide, such as an IgG Fcfusion region peptide or leader or secretory sequence or a sequencewhich is employed for purification of the above form of the polypeptideor a proprotein sequence. Such fragments, derivatives and analogs aredeemed to be within the scope of those skilled in the art from theteachings herein.

Thus, the B. burgdorferi polypeptides of the present invention mayinclude one or more amino acid substitutions, deletions, or additions,either from natural mutations or human manipulation. As indicated,changes are preferably of a minor nature, such as conservative aminoacid substitutions that do not significantly affect the folding oractivity of the protein (see Table 3).

Amino acids in the B. burgdorferi proteins of the present invention thatare essential for function can be identified by methods known in theart, such as site-directed mutagenesis or alanine-scanning mutagenesis.See, e.g., Cunningham et al. (1989) Science 244: 1081-1085. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity using assays appropriate for measuring the function of theparticular protein.

Of special interest are substitutions of charged amino acids with othercharged or neutral amino acids which may produce proteins with highlydesirable improved characteristics, such as less aggregation.Aggregation may not only reduce activity but also be problematic whenpreparing pharmaceutical formulations, because aggregates can beimmunogenic. See, e.g., Pinckard et al., (1967) Clin. Exp. Immunol. 2:331-340; Robbins, et al., (1987) Diabetes 36: 838-845; Cleland, et al.,(1993) Crit. Rev. Therapeutic Drug Carrier Systems 10: 307-377.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Arecombinantly produced version of the B. burgdorferi polypeptide can besubstantially purified by the one-step method described by Smith et al.(1988) Gene 67: 31-40. Polypeptides of the invention also can bepurified from natural or recombinant sources using antibodies directedagainst the polypeptides of the invention in methods which are wellknown in the art of protein purification.

The invention further provides for isolated B. burgdorferi polypeptidescomprising an amino acid sequence selected from the group consisting of:(a) the amino acid sequence of a full-length B. burgdorferi polypeptidehaving the complete amino acid sequence shown in Table 1; (b) the aminoacid sequence of a full-length B. burgdorferi polypeptide having thecomplete amino acid sequence shown in Table 1 excepting the N-terminalmethionine; (c) the complete amino acid sequence encoded by the plasmidslisted in Table 1; and (d) the complete amino acid sequence exceptingthe N-terminal methionine encoded by the plasmids listed in Table 1. Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least90% identical, and still more preferably 95%, 96%, 97%, 98% or 99%identical to those described in (a), (b), (c), and (d) above.

Further polypeptides of the present invention include polypeptides whichhave at least 90% similarity, more preferably at least 95% similarity,and still more preferably at least 96%, 97%, 98% or 99% similarity tothose described above.

A further embodiment of the invention relates to a polypeptide whichcomprises the amino acid sequence of a B. burgdorferi polypeptide havingan amino acid sequence which contains at least one conservative aminoacid substitution, but not more than 50 conservative amino acidsubstitutions, not more than 40 conservative amino acid substitutions,not more than 30 conservative amino acid substitutions, and not morethan 20 conservative amino acid substitutions. Also provided arepolypeptides which comprise the amino acid sequence of a B. burgdorferipolypeptide, having at least one, but not more than 10, 9, 8, 7, 6, 5,4, 3, 2 or 1 conservative amino acid substitutions.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequences shown in Table 1 or to the amino acid sequence encoded bythe plasmids listed in Table 1 can be determined conventionally usingknown computer programs. A preferred method for determining the bestoverall match between a query sequence (a sequence of the presentinvention) and a subject sequence, also referred to as a global sequencealignment, can be determined using the FASTDB computer program based onthe algorithm of Brutlag et al., (1990) Comp. App. Biosci. 6: 237-245.In a sequence alignment the query and subject sequences are both aminoacid sequences. The result of said global sequence alignment is inpercent identity. Preferred parameters used in a FASTDB amino acidalignment are: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, JoiningPenalty=20, Randomization Group Length=0, Cutoff Score=1, WindowSize=sequence length, Gap Penalty=5, Gap Size Penalty=0.05, WindowSize=500 or the length of the subject amino acid sequence, whichever isshorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, the results, inpercent identity, must be manually corrected. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query amino acid residues outside the farthest N-and C-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not match/align with the first 10 residues at theN-terminus. The 10 unpaired residues represent 10% of the sequence(number of residues at the N- and C-termini not matched/total number ofresidues in the query sequence) so 10% is subtracted from the percentidentity score calculated by the FASTDB program. If the remaining 90residues were perfectly matched the final percent identity would be 90%.In another example, a 90 residue subject sequence is compared with a 100residue query sequence. This time the deletions are internal so thereare no residues at the N- or C-termini of the subject sequence which arenot matched/aligned with the query. In this case the percent identitycalculated by FASTDB is not manually corrected. Once again, only residuepositions outside the N- and C-terminal ends of the subject sequence, asdisplayed in the FASTDB alignment, which are not matched/aligned withthe query sequence are manually corrected. No other manual correctionsare to made for the purposes of the present invention.

The above polypeptide sequences are included irrespective of whetherthey have their normal biological activity. This is because even where aparticular polypeptide molecule does not have biological activity, oneof skill in the art would still know how to use the polypeptide, forinstance, as a vaccine or to generate antibodies. Other uses of thepolypeptides of the present invention that do not have B. burgdorferiactivity include, inter alia, as epitope tags, in epitope mapping, andas molecular weight markers on SDS-PAGE gels or on molecular sieve gelfiltration columns using methods known to those of skill in the art.

As described below, the polypeptides of the present invention can alsobe used to raise polyclonal and monoclonal antibodies, which are usefulin assays for detecting B. burgdorferi protein expression or as agonistsand antagonists capable of enhancing or inhibiting B. burgdorferiprotein function. Further, such polypeptides can be used in the yeasttwo-hybrid system to “capture” B. burgdorferi protein binding proteinswhich are also candidate agonists and antagonists according to thepresent invention. See, e.g., Fields et al. (1989) Nature 340: 245-246.

Epitope-Bearing Portions

In another aspect, the invention provides peptides and polypeptidescomprising epitope-bearing portions of the B. burgdorferi polypeptidesof the present invention. These epitopes are immunogenic or antigenicepitopes of the polypeptides of the present invention. An “immunogenicepitope” is defined as a part of a protein that elicits an antibodyresponse when the whole protein or polypeptide is the immunogen. Theseimmunogenic epitopes are believed to be confined to a few loci on themolecule. On the other hand, a region of a protein molecule to which anantibody can bind is defined as an “antigenic determinant” or “antigenicepitope.” The number of immunogenic epitopes of a protein generally isless than the number of antigenic epitopes. See, e.g., Geysen, et al.(1983) Proc. Natl. Acad. Sci. USA 81: 3998-4002. Predicted antigenicepitopes are shown in Table 4, below. It is pointed out that Table 4only lists amino acid residues comprising epitopes predicted to have thehighest degree of antigenicity. The polypeptides not listed in Table 4and portions of polypeptides not listed in Table 4 are not considerednon-antigenic. This is because they may still be antigenic in vivo butmerely not recognized as such by the particular algorithm used. Thus,Table 4 lists the amino acid residues comprising preferred antigenicepitopes but not a complete list. Amino acid residues comprising otherantigenic epitopes may be determined by algorithms similar to theJameson-Wolf analysis or by in vivo testing for an antigenic responseusing the methods described herein or those known in the art.

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, e.g., Sutcliffe, et al., (1983) Science 219:660-666. Peptides capable of eliciting protein-reactive sera arefrequently represented in the primary sequence of a protein, can becharacterized by a set of simple chemical rules, and are confinedneither to immunodominant regions of intact proteins (i.e., immunogenicepitopes) nor to the amino or carboxyl terminals. Peptides that areextremely hydrophobic and those of six or fewer residues generally areineffective at inducing antibodies that bind to the mimicked protein;longer, peptides, especially those containing proline residues, usuallyare effective. See, Sutcliffe, et al., supra, p. 661. For instance, 18of 20 peptides designed according to these guidelines, containing 8-39residues covering 75% of the sequence of the influenza virushemagglutinin HA1 polypeptide chain, induced antibodies that reactedwith the HA1 protein or intact virus; and 12/12 peptides from the MuLVpolymerase and 18/18 from the rabies glycoprotein induced antibodiesthat precipitated the respective proteins.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. Thus, a highproportion of hybridomas obtained by fusion of spleen cells from donorsimmunized with an antigen epitope-bearing peptide generally secreteantibody reactive with the native protein. See Sutcliffe, et al., supra,p. 663. The antibodies raised by antigenic epitope-bearing peptides orpolypeptides are useful to detect the mimicked protein, and antibodiesto different peptides may be used for tracking the fate of variousregions of a protein precursor which undergoes post-translationalprocessing. The peptides and anti-peptide antibodies may be used in avariety of qualitative or quantitative assays for the mimicked protein,for instance in competition assays since it has been shown that evenshort peptides (e.g., about 9 amino acids) can bind and displace thelarger peptides in immunoprecipitation assays. See, e.g., Wilson, etal., (1984) Cell 37: 767-778. The anti-peptide antibodies of theinvention also are useful for purification of the mimicked protein, forinstance, by adsorption chromatography using methods known in the art.

Antigenic epitope-bearing peptides and polypeptides of the inventiondesigned according to the above guidelines preferably contain a sequenceof at least seven, more preferably at least nine and most preferablybetween about 10 to about 50 amino acids (i.e. any integer between 7 and50) contained within the amino acid sequence of a polypeptide of theinvention. However, peptides or polypeptides comprising a larger portionof an amino acid sequence of a polypeptide of the invention, containingabout 50 to about 100 amino acids, or any length up to and including theentire amino acid sequence of a polypeptide of the invention, also areconsidered epitope-bearing peptides or polypeptides of the invention andalso are useful for inducing antibodies that react with the mimickedprotein. Preferably, the amino acid sequence of the epitope-bearingpeptide is selected to provide substantial solubility in aqueoussolvents (i.e., the sequence includes relatively hydrophilic residuesand highly hydrophobic sequences are preferably avoided); and sequencescontaining proline residues are particularly preferred.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate an Borrelia-specific immune response or antibodiesinclude portions of the amino acid sequences identified in Table 1. Morespecifically, Table 4 discloses a list of non-limiting residues that areinvolved in the antigenicity of the epitope-bearing fragments of thepresent invention. Therefore, the present inventions provides forisolated and purified antigenic epitope-bearing fragments of thepolypeptides of the present invention comprising a peptide sequences ofTable 4. The antigenic epitope-bearing fragments comprising a peptidesequence of Table 4 preferably contain a sequence of at least seven,more preferably at least nine and most preferably between about 10 toabout 50 amino acids (i.e. any integer between 7 and 50) of apolypeptide of the present invention. That is, included in the presentinvention are antigenic polypeptides between the integers of 7 and 50amino acid in length comprising one or more of the sequences of Table 4.Therefore, in most cases, the polypeptides of Table 4 make up only aportion of the antigenic polypeptide. All combinations of sequencesbetween the integers of 7 and 50 amino acid in length comprising one ormore of the sequences of Table 4 are included. The antigenicepitope-bearing fragments may be specified by either the number ofcontiguous amino acid residues or by specific N-terminal and C-terminalpositions as described above for the polypeptide fragments of thepresent invention, wherein the initiation codon is residue 1. Any numberof the described antigenic epitope-bearing fragments of the presentinvention may also be excluded from the present invention in the samemanner.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means for making peptides or polypeptidesincluding recombinant means using nucleic acid molecules of theinvention. For instance, an epitope-bearing amino acid sequence of thepresent invention may be fused to a larger polypeptide which acts as acarrier during recombinant production and purification, as well asduring immunization to produce anti-peptide antibodies. Epitope-bearingpeptides also may be synthesized using known methods of chemicalsynthesis. For instance, Houghten has described a simple method forsynthesis of large numbers of peptides, such as 10-20 mg of 248different 13 residue peptides representing single amino acid variants ofa segment of the HA1 polypeptide which were prepared and characterized(by ELISA-type binding studies) in less than four weeks (Houghten, R. A.Proc. Natl. Acad. Sci. USA 82: 5131-5135 (1985)). This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten and coworkers (1986). In this procedurethe individual resins for the solid-phase synthesis of various peptidesare contained in separate solvent-permeable packets, enabling theoptimal use of the many identical repetitive steps involved insolid-phase methods. A completely manual procedure allows 500-1000 ormore syntheses to be conducted simultaneously (Houghten et al. (1985)Proc. Natl. Acad. Sci. 82: 5131-5135 at 5134.

Epitope-bearing peptides and polypeptides of the invention are used toinduce antibodies according to methods well known in the art. See, e.g.,Sutcliffe, et al., supra; Wilson, et al., supra; and Bittle, et al.(1985) J. Gen. Virol. 66: 2347-2354. Generally, animals may be immunizedwith free peptide; however, anti-peptide antibody titer may be boostedby coupling of the peptide to a macromolecular carrier, such as keyholelimpet hemacyanin (KLH) or tetanus toxoid. For instance, peptidescontaining cysteine may be coupled to carrier using a linker such asm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while otherpeptides may be coupled to carrier using a more general linking agentsuch as glutaraldehyde. Animals such as rabbits, rats and mice areimmunized with either free or carrier-coupled peptides, for instance, byintraperitoneal and/or intradermal injection of emulsions containingabout 100 μg peptide or carrier protein and Freund's adjuvant. Severalbooster injections may be needed, for instance, at intervals of abouttwo weeks, to provide a useful titer of anti-peptide antibody which canbe detected, for example, by ELISA assay using free peptide adsorbed toa solid surface. The titer of anti-peptide antibodies in serum from animmunized animal may be increased by selection of anti-peptideantibodies, for instance, by adsorption to the peptide on a solidsupport and elution of the selected antibodies according to methods wellknown in the art.

Immunogenic epitope-bearing peptides of the invention, i.e., those partsof a protein that elicit an antibody response when the whole protein isthe immunogen, are identified according to methods known in the art. Forinstance, Geysen, et al., supra, discloses a procedure for rapidconcurrent synthesis on solid supports of hundreds of peptides ofsufficient purity to react in an ELISA. Interaction of synthesizedpeptides with antibodies is then easily detected without removing themfrom the support. In this manner a peptide bearing an immunogenicepitope of a desired protein may be identified routinely by one ofordinary skill in the art. For instance, the immunologically importantepitope in the coat protein of foot-and-mouth disease virus was locatedby Geysen et al. supra with a resolution of seven amino acids bysynthesis of an overlapping set of all 208 possible hexapeptidescovering the entire 213 amino acid sequence of the protein. Then, acomplete replacement set of peptides in which all 20 amino acids weresubstituted in turn at every position within the epitope weresynthesized, and the particular amino acids conferring specificity forthe reaction with antibody were determined. Thus, peptide analogs of theepitope-bearing peptides of the invention can be made routinely by thismethod. U.S. Pat. No. 4,708,781 to Geysen (1987) further describes thismethod of identifying a peptide bearing an immunogenic epitope of adesired protein.

Further still, U.S. Pat. No. 5,194,392, to Geysen (1990), describes ageneral method of detecting or determining the sequence of monomers(amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a “mimotope”) which is complementary to a particularparatope (antigen binding site) of an antibody of interest. Moregenerally, U.S. Pat. No. 4,433,092, also to Geysen (1989), describes amethod of detecting or determining a sequence of monomers which is atopographical equivalent of a ligand which is complementary to theligand binding site of a particular receptor of interest. Similarly,U.S. Pat. No. 5,480,971 to Houghten, R. A. et al. (1996) discloseslinear C₁-C₇-alkyl peralkylated oligopeptides and sets and libraries ofsuch peptides, as well as methods for using such oligopeptide sets andlibraries for determining the sequence of a peralkylated oligopeptidethat preferentially binds to an acceptor molecule of interest. Thus,non-peptide analogs of the epitope-bearing peptides of the inventionalso can be made routinely by these methods. The entire disclosure ofeach document cited in this section on “Polypeptides and Fragments” ishereby incorporated herein by reference.

As one of skill in the art will appreciate, the polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins. (EPA 0,394,827; Traunecker et al. (1988) Nature 331:84-86. Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than a monomeric B. burgdorferi polypeptideor fragment thereof alone. See Fountoulakis et al. (1995) J. Biochem.270: 3958-3964. Nucleic acids encoding the above epitopes of B.burgdorferi polypeptides can also be recombined with a gene of interestas an epitope tag to aid in detection and purification of the expressedpolypeptide.

Antibodies

B. burgdorferi protein-specific antibodies for use in the presentinvention can be raised against the intact B. burgdorferi protein or anantigenic polypeptide fragment thereof, which may be presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules, single chain whole antibodies, andantibody fragments. Antibody fragments of the present invention includeFab and F(ab′)2 and other fragments including single-chain Fvs (scFv)and disulfide-linked Fvs (sdFv). Also included in the present inventionare chimeric and humanized monoclonal antibodies and polyclonalantibodies specific for the polypeptides of the present invention. Theantibodies of the present invention may be prepared by any of a varietyof methods. For example, cells expressing a polypeptide of the presentinvention or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. For example, a preparation of B. burgdorferi polypeptide orfragment thereof is prepared and purified to render it substantiallyfree of natural contaminants. Such a preparation is then introduced intoan animal in order to produce polyclonal antisera of greater specificactivity.

In a preferred method, the antibodies of the present invention aremonoclonal antibodies or binding fragments thereof. Such monoclonalantibodies can be prepared using hybridoma technology. See, e.g., Harlowet al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor LaboratoryPress, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES ANDT-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981). Fab and F(ab)2fragments may be produced by proteolytic cleavage, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab)2fragments). Alternatively, B. burgdorferi polypeptide-binding fragments,chimeric, and humanized antibodies can be produced through theapplication of recombinant DNA technology or through synthetic chemistryusing methods known in the art.

Alternatively, additional antibodies capable of binding to thepolypeptide antigen of the present invention may be produced in atwo-step procedure through the use of anti-idiotypic antibodies. Such amethod makes use of the fact that antibodies are themselves antigens,and that, therefore, it is possible to obtain an antibody which binds toa second antibody. In accordance with this method, B. burgdorferipolypeptide-specific antibodies are used to immunize an animal,preferably a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones which produce an antibody whose ability to bind to theB. burgdorferi polypeptide-specific antibody can be blocked by the B.burgdorferi polypeptide antigen. Such antibodies comprise anti-idiotypicantibodies to the B. burgdorferi polypeptide-specific antibody and canbe used to immunize an animal to induce formation of further B.burgdorferi polypeptide-specific antibodies.

Antibodies and fragments thereof of the present invention may bedescribed by the portion of a polypeptide of the present inventionrecognized or specifically bound by the antibody. Antibody bindingfragments of a polypeptide of the present invention may be described orspecified in the same manner as for polypeptide fragments discussedabove., i.e., by N-terminal and C-terminal positions or by size incontiguous amino acid residues. Any number of antibody bindingfragments, of a polypeptide of the present invention, specified byN-terminal and C-terminal positions or by size in amino acid residues,as described above, may also be excluded from the present invention.Therefore, the present invention includes antibodies the specificallybind a particularly described fragment of a polypeptide of the presentinvention and allows for the exclusion of the same.

Antibodies and fragments thereof of the present invention may also bedescribed or specified in terms of their cross-reactivity. Antibodiesand fragments that do not bind polypeptides of any other species ofBorrelia other than B. burgdorferi are included in the presentinvention. Likewise, antibodies and fragments that bind only species ofBorrelia, i.e. antibodies and fragments that do not bind bacteria fromany genus other than Borrelia, are included in the present invention.

Diagnostic Assays

The present invention further relates to methods for assayingstaphylococcal infection in an animal by detecting the expression ofgenes encoding staphylococcal polypeptides of the present invention. Themethods comprise analyzing tissue or body fluid from the animal forBorrelia-specific antibodies, nucleic acids, or proteins. Analysis ofnucleic acid specific to Borrelia is assayed by PCR or hybridizationtechniques using nucleic acid sequences of the present invention aseither hybridization probes or primers. See, e.g., Sambrook et al.Molecular cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, 2nd ed., 1989, page 54 reference); Eremeeva et al. (1994) J.Clin. Microbiol. 32: 803-810 (describing differentiation among spottedfever group Rickettsiae species by analysis of restriction fragmentlength polymorphism of PCR-amplified DNA) and Chen et al. 1994 J. Clin.Microbiol. 32: 589-595 (detecting B. burgdorferi nucleic acids via PCR).

Where diagnosis of a disease state related to infection with Borreliahas already been lade, the present invention is useful for monitoringprogression or regression of the disease state whereby patientsexhibiting enhanced Borrelia gene expression will experience a worseclinical outcome relative to patients expressing these gene(s) at alower level.

By “biological sample” is intended any biological sample obtained froman animal, cell line, tissue culture, or other source which containsBorrelia polypeptide, mRNA, or DNA. Biological samples include bodyfluids (such as saliva, blood, plasma, urine, mucus, synovial fluid,etc.) tissues (such as muscle, skin, and cartilage) and any otherbiological source suspected of containing Borrelia polypeptides ornucleic acids. Methods for obtaining biological samples such as tissueare well known in the art.

The present invention is useful for detecting diseases related toBorrelia infections in animals. Preferred animals include monkeys, apes,cats, dogs, birds, cows, pigs, mice, horses, rabbits and humans.Particularly preferred are humans.

Total RNA can be isolated from a biological sample using any suitabletechnique such as the single-stepguanidinium-thiocyanate-phenol-chloroform method described inChomczynski et al. (1987) Anal. Biochem. 162: 156-159. mRNA encodingBorrelia polypeptides having sufficient homology to the nucleic acidsequences identified in Table 1 to allow for hybridization betweencomplementary sequences are then assayed using any appropriate method.These include Northern blot analysis, S1 nuclease mapping, thepolymerase chain reaction (PCR), reverse transcription in combinationwith the polymerase chain reaction (RT-PCR), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.(1990) Cell 63: 303-312. Briefly, total RNA is prepared from abiological sample as described above. For the Northern blot, the RNA isdenatured in an appropriate buffer (such as glyoxal/dimethylsulfoxide/sodium phosphate buffer), subjected to agarose gelelectrophoresis, and transferred onto a nitrocellulose filter. After theRNAs have been linked to the filter by a UV linker, the filter isprehybridized in a solution containing formamide, SSC, Denhardt'ssolution, denatured salmon sperm, SDS, and sodium phosphate buffer. A B.burgdorferi polynucleotide sequence shown in Table 1 labeled accordingto any appropriate method (such as the ³²P-multiprimed DNA labelingsystem (Amersham)) is used as probe. After hybridization overnight, thefilter is washed and exposed to x-ray film. DNA for use as probeaccording to the present invention is described in the sections aboveand will preferably at least 15 nucleotides in length.

S1 mapping can be performed as described in Fujita et al. (1987) Cell49: 357-367. To prepare probe DNA for use in S1 mapping, the sensestrand of an above-described B. burgdorferi DNA sequence of the presentinvention is used as a template to synthesize labeled antisense DNA. Theantisense DNA can then be digested using an appropriate restrictionendonuclease to generate further DNA probes of a desired length. Suchantisense probes are useful for visualizing protected bandscorresponding to the target mRNA (i.e., mRNA encoding Borreliapolypeptides).

Levels of mRNA encoding Borrelia polypeptides are assayed, for e.g.,using the RT-PCR method described in Makino et al. (1990) Technique 2:295-301. By this method, the radioactivities of the “amplicons” in thepolyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding the Borreliapolypeptides of the present invention) are quantified using an imaginganalyzer. RT and PCR reaction ingredients and conditions, reagent andgel concentrations, and labeling methods are well known in the art.Variations on the RT-PCR method will be apparent to the skilled artisan.Other PCR methods that can detect the nucleic acid of the presentinvention can be found in PCR PRIMER: A LABORATORY MANUAL (C. W.Dieffenbach et al. eds., Cold Spring Harbor Lab Press, 1995).

The polynucleotides of the present invention, including both DNA andRNA, may be used to detect polynucleotides of the present invention orBorrelia species including B. burgdorferi using bio chip technology. Thepresent invention includes both high density chip arrays (>1000oligonucleotides per cm²) and low density chip arrays (<1000oligonucleotides per cm²). Bio chips comprising arrays ofpolynucleotides of the present invention may be used to detect Borreliaspecies, including B. burgdorferi, in biological and environmentalsamples and to diagnose an animal, including humans, with an B.burgdorferi or other Borrelia infection. The bio chips of the presentinvention may comprise polynucleotide sequences of other pathogensincluding bacteria, viral, parasitic, and fungal polynucleotidesequences, in addition to the polynucleotide sequences of the presentinvention, for use in rapid differential pathogenic detection anddiagnosis. The bio chips can also be used to monitor an B. burgdorferior other Borrelia infections and to monitor the genetic changes(deletions, insertions, mismatches, etc.) in response to drug therapy inthe clinic and drug development in the laboratory. The bio chiptechnology comprising arrays of polynucleotides of the present inventionmay also be used to simultaneously monitor the expression of amultiplicity of genes, including those of the present invention. Thepolynucleotides used to comprise a selected array may be specified inthe same manner as for the fragments, i.e., by their 5′ and 3′ positionsor length in contiguous base pairs and include from. Methods andparticular uses of the polynucleotides of the present invention todetect Borrelia species, including B. burgdorferi, using bio chiptechnology include those known in the art and those of: U.S. Pat. Nos.5,510,270, 5,545,531, 5,445,934, 5,677,195, 5,532,128, 5,556,752,5,527,681, 5,451,683, 5,424,186, 5,60,7646, 5,658,732 and World PatentNos. WO/9710365, WO/9511995, WO/9743447, WO/9535505, each incorporatedherein in their entireties.

Biosensors using the polynucleotides of the present invention may alsobe used to detect, diagnose, and monitor B. burgdorferi or otherBorrelia species and infections thereof. Biosensors using thepolynucleotides of the present invention may also be used to detectparticular polynucleotides of the present invention. Biosensors usingthe polynucleotides of the present invention may also be used to monitorthe genetic changes (deletions, insertions, mismatches, etc.) inresponse to drug therapy in the clinic and drug development in thelaboratory. Methods and particular uses of the polynucleotides of thepresent invention to detect Borrelia species, including B. burgdorferi,using biosensors include those known in the art and those of: U.S. Pat.Nos. 5,721,102, 5,658,732, 5631170, and World Patent Nos. WO97/35011,WO/9720203, each incorporated herein in their entireties.

Thus, the present invention includes both bio chips and biosensorscomprising polynucleotides of the present invention and methods of theiruse.

Assaying Borrelia polypeptide levels in a biological sample can occurusing any art-known method, such as antibody-based techniques. Forexample, Borrelia polypeptide expression in tissues can be studied withclassical immunohistological methods. In these, the specific recognitionis provided by the primary antibody (polyclonal or monoclonal) but thesecondary detection system can utilize fluorescent, enzyme, or otherconjugated secondary antibodies. As a result, an immunohistologicalstaining of tissue section for pathological examination is obtained.Tissues can also be extracted, e.g., with urea and neutral detergent,for the liberation of Borrelia polypeptides for Western-blot or dot/slotassay. See, e.g., Jalkanen, M. et al. (1985) J. Cell. Biol. 101:976-985; Jalkanen, M. et al. (1987) J. Cell. Biol. 105: 3087-3096. Inthis technique, which is based on the use of cationic solid phases,quantitation of a Borrelia polypeptide can be accomplished using anisolated Borrelia polypeptide as a standard. This technique can also beapplied to body fluids.

Other antibody-based methods useful for detecting Borrelia polypeptidegene expression include immunoassays, such as the ELISA and theradioimmunoassay (RIA). For example, a Borrelia polypeptide-specificmonoclonal antibodies can be used both as an immunoabsorbent and as anenzyme-labeled probe to detect and quantify a Borrelia polypeptide. Theamount of a Borrelia polypeptide present in the sample can be calculatedby reference to the amount present in a standard preparation using alinear regression computer algorithm. Such an ELISA is described inIacobelli et al. (1988) Breast Cancer Research and Treatment 11: 19-30.In another ELISA assay, two distinct specific monoclonal antibodies canbe used to detect Borrelia polypeptides in a body fluid. In this assay,one of the antibodies is used as the immunoabsorbent and the other asthe enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contacting the Borreliapolypeptide with immobilized antibody and, without washing, contactingthe mixture with the labeled antibody. The “two-step” assay involveswashing before contacting the mixture with the labeled antibody. Otherconventional methods may also be employed as suitable. It is usuallydesirable to immobilize one component of the assay system on a support,thereby allowing other components of the system to be brought intocontact with the component and readily removed from the sample.Variations of the above and other immunological methods included in thepresent invention can also be found in Harlow et al., ANTIBODIES: ALABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988).

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

Further suitable labels for the Borrelia polypeptide-specific antibodiesof the present invention are provided below. Examples of suitable enzymelabels include malate dehydrogenase, Borrelia nuclease, delta-5-steroidisomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphatedehydrogenase, triose phosphate isomerase, peroxidase, alkalinephosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging. See, e.g., Perkins et al. (1985) Eur. J. Nucl. Med. 10:296-301; Carasquillo et al. (1987) J. Nucl. Med. 28: 281-287. Forexample, ¹¹¹In coupled to monoclonal antibodies with1-(P-isothiocyanatobenzyl)-DPTA has shown little uptake in non-tumorstissues, particularly the liver, and therefore enhances specificity oftumor localization. See, Esteban et al. (1987) J. Nucl. Med. 28:861-870.

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include, Pseudomonas toxin, diphtheriatoxin, ricin, and cholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al. (1976) Clin. Chim. Acta 70: 1-31, andSchurs et al. (1977) Clin. Chim. Acta 81: 1-40. Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

In a related aspect, the invention includes a diagnostic kit for use inscreening serum containing antibodies specific against B. burgdorferiinfection. Such a kit may include an isolated B. burgdorferi antigencomprising an epitope which is specifically immunoreactive with at leastone anti-B. burgdorferi antibody. Such a kit also includes means fordetecting the binding of said antibody to the antigen. In specificembodiments, the kit may include a recombinantly produced or chemicallysynthesized peptide or polypeptide antigen. The peptide or polypeptideantigen may be attached to a solid support.

In a more specific embodiment, the detecting means of theabove-described kit includes a solid support to which said peptide orpolypeptide antigen is attached. Such a kit may also include anon-attached reporter-labeled anti-human antibody. In this embodiment,binding of the antibody to the B. burgdorferi antigen can be detected bybinding of the reporter labeled antibody to the anti-B. burgdorferipolypeptide antibody.

In a related aspect, the invention includes a method of detecting B.burgdorferi infection in a subject. This detection method includesreacting a body fluid, preferably serum, from the subject with anisolated B. burgdorferi antigen, and examining the antigen for thepresence of bound antibody. In a specific embodiment, the methodincludes a polypeptide antigen attached to a solid support, and serum isreacted with the support. Subsequently, the support is reacted with areporter-labeled anti-human antibody. The support is then examined forthe presence of reporter-labeled antibody.

The solid surface reagent employed in the above assays and kits isprepared by known techniques for attaching protein material to solidsupport material, such as polymeric beads, dip sticks, 96-well plates orfilter material. These attachment methods generally include non-specificadsorption of the protein to the support or covalent attachment of theprotein, typically through a free amine group, to a chemically reactivegroup on the solid support, such as an activated carboxyl, hydroxyl, oraldehyde group. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

The polypeptides and antibodies of the present invention, includingfragments thereof, may be used to detect Borrelia species including B.burgdorferi using bio chip and biosensor technology. Bio chip andbiosensors of the present invention may comprise the polypeptides of thepresent invention to detect antibodies, which specifically recognizeBorrelia species, including B. burgdorferi. Bio chip and biosensors ofthe present invention may also comprise antibodies which specificallyrecognize the polypeptides of the present invention to detect Borreliaspecies, including B. burgdorferi or specific polypeptides of thepresent invention. Bio chips or biosensors comprising polypeptides orantibodies of the present invention may be used to detect Borreliaspecies, including B. burgdorferi, in biological and environmentalsamples and to diagnose an animal, including humans, with an B.burgdorferi or other Borrelia infection. Thus, the present inventionincludes both bio chips and biosensors comprising polypeptides orantibodies of the present invention and methods of their use.

The bio chips of the present invention may further comprise polypeptidesequences of other pathogens including bacteria, viral, parasitic, andfungal polypeptide sequences, in addition to the polypeptide sequencesof the present invention, for use in rapid differential pathogenicdetection and diagnosis. The bio chips of the present invention mayfurther comprise antibodies or fragments thereof specific for otherpathogens including bacteria, viral, parasitic, and fungal polypeptidesequences, in addition to the antibodies or fragments thereof of thepresent invention, for use in rapid differential pathogenic detectionand diagnosis. The bio chips and biosensors of the present invention mayalso be used to monitor an B. burgdorferi or other Borrelia infectionand to monitor the genetic changes (amino acid deletions, insertions,substitutions, etc.) in response to drug therapy in the clinic and drugdevelopment in the laboratory. The bio chip and biosensors comprisingpolypeptides or antibodies of the present invention may also be used tosimultaneously monitor the expression of a multiplicity of polypeptides,including those of the present invention. The polypeptides used tocomprise a bio chip or biosensor of the present invention may bespecified in the same manner as for the fragments, i.e., by theirN-terminal and C-terminal positions or length in contiguous amino acidresidue. Methods and particular uses of the polypeptides and antibodiesof the present invention to detect Borrelia species, including B.burgdorferi, or specific polypeptides using bio chip and biosensortechnology include those known in the art, those of the U.S. Patent Nos.and World Patent Nos. listed above for bio chips and biosensors usingpolynucleotides of the present invention, and those of: U.S. Pat. Nos.5,658,732, 5,135,852, 5567301, 5,677,196, 5,690,894 and World PatentNos. WO9729366, WO9612957, each incorporated herein in their entireties.

Treatment:

Agonists and Antagonists—Assays and Molecules

The invention also provides a method of screening compounds to identifythose which enhance or block the biological activity of the B.burgdorferi polypeptides of the present invention. The present inventionfurther provides where the compounds kill or slow the growth of B.burgdorferi. The ability of B. burgdorferi antagonists, including B.burgdorferi ligands, to prophylactically or therapeutically blockantibiotic resistance may be easily tested by the skilled artisan. See,e.g., Straden et al. (1997) J. Bacteriol. 179(1): 9-16.

An agonist is a compound which increases the natural biological functionor which functions in a manner similar to the polypeptides of thepresent invention, while antagonists decrease or eliminate suchfunctions. Potential antagonists include small organic molecules,peptides, polypeptides, and antibodies that bind to a polypeptide of theinvention and thereby inhibit or extinguish its activity.

The antagonists may be employed for instance to inhibit peptidoglycancross bridge formation. Antibodies against B. burgdorferi may beemployed to bind to and inhibit B. burgdorferi activity to treatantibiotic resistance. Any of the above antagonists may be employed in acomposition with a pharmaceutically acceptable carrier.

Vaccines

The present invention also provides vaccines comprising one or morepolypeptides of the present invention. Heterogeneity in the compositionof a vaccine may be provided by combining B. burgdorferi polypeptides ofthe present invention. Multi-component vaccines of this type aredesirable because they are likely to be more effective in elicitingprotective immune responses against multiple species and strains of theBorrelia genus than single polypeptide vaccines. Thus, as discussed indetail below, a multi-component vaccine of the present invention maycontain one or more, preferably 2 to about 20, more preferably 2 toabout 15, and most preferably 3 to about 8, of the B. burgdorferipolypeptides shown in Table 1, or fragments thereof.

Multi-component vaccines are known in the art to elicit antibodyproduction to numerous immunogenic components. Decker, M. and Edwards,K., J. Infect. Dis. 174: S270-275 (1996). In addition, a hepatitis B,diphtheria, tetanus, pertussis tetravalent vaccine has recently beendemonstrated to elicit protective levels of antibodies in human infantsagainst all four pathogenic agents. Aristegui, J. et al., Vaccine 15:7-9 (1997).

The present invention thus also includes multi-component vaccines. Thesevaccines comprise more than one polypeptide, immunogen or antigen. Anexample of such a multi-component vaccine would be a vaccine comprisingmore than one of the B. burgdorferi polypeptides shown in Table 1. Asecond example is a vaccine comprising one or more, for example 2 to 10,of the B. burgdorferi polypeptides shown in Table 1 and one or more, forexample 2 to 10, additional polypeptides of either borrelial ornon-borrelial origin. Thus, a multi-component vaccine which confersprotective immunity to both a borrelial infection and infection byanother pathogenic agent is also within the scope of the invention.

As indicated above, the vaccines of the present invention are expectedto elicit a protective immune response against infections caused byspecies and strains of Borrelia other than B. burgdorferi sensu strictoisolate B31 (ATCC Accession No. 35210). Immunizations usingdecorin-binding protein and OspA derived from one strain of B.burgdorferi has been shown to elicit the production of antiserum whichconfers passive immunity against other strains of B. burgdorferi.Cassatt, D. et al., Protection of Borrelia burgdorferi Infection byAntibodies to Decorin-binding Protein, in VACCINES97, Cold Spring HarborPress (1997), pages 191-195. Further, the inventors have found using anin vitro assay that antiserum produced in response to B. burgdorferidecorin-binding protein will kill several species of Borrelia. The aminoacid sequences of decorin-binding protein expressed by different strainsof B. burgdorferi are believed to diverge by as much as 25%. Thus,antisera elicited against decorin-binding proteins confers passiveimmunity against Borrelia expressing proteins having only 75% or lessamino acid sequence similarity.

Further within the scope of the invention are whole cell and whole viralvaccines. Such vaccines may be produced recombinantly and involve theexpression of one or more of the B. burgdorferi polypeptides shown inTable 1. For example, the B. burgdorferi polypeptides of the presentinvention may be either secreted or localized intracellular, on the cellsurface, or in the periplasmic space. Further, when a recombinant virusis used, the B. burgdorferi polypeptides of the present invention may,for example, be localized in the viral envelope, on the surface of thecapsid, or internally within the capsid. Whole cells vaccines whichemploy cells expressing heterologous proteins are known in the art. See,e.g., Robinson, K. et al., Nature Biotech. 15: 653-657 (1997); Sirard,J. et al., Infect. Immun. 65: 2029-2033 (1997); Chabalgoity, J. et al.,Infect. Immun. 65: 2402-2412 (1997). These cells may be administeredlive or may be killed prior to administration. Chabalgoity, J. et al.,supra, for example, report the successful use in mice of a liveattenuated Salmonella vaccine strain which expresses a portion of aplatyhelminth fatty acid-binding protein as a fusion protein on itscells surface.

A multi-component vaccine can also be prepared using techniques known inthe art by combining one or more B. burgdorferi polypeptides of thepresent invention, or fragments thereof, with additional non-borrelialcomponents (e.g., diphtheria toxin or tetanus toxin, and/or othercompounds known to elicit an immune response). Such vaccines are usefulfor eliciting protective immune responses to both members of theBorrelia genus and non-borrelial pathogenic agents.

The vaccines of the present invention also include DNA vaccines. DNAvaccines are currently being developed for a number of infectiousdiseases. Boyer, J et al., Nat. Med. 3: 526-532 (1997); reviewed inSpier, R., Vaccine 14: 1285-1288 (1996). Such DNA vaccines contain anucleotide sequence encoding one or more B. burgdorferi polypeptides ofthe present invention oriented in a manner that allows for expression ofthe subject polypeptide. The direct administration of plasmid DNAencoding OspA has been shown to elicit protective immunity in miceagainst borrelial challenge. Luke, C. et al., J. Infect. Dis. 175: 91-97(1997).

The present invention also relates to the administration of a vaccinewhich is co-administered with a molecule capable of modulating immuneresponses. Kim, J. et al., Nature Biotech. 15: 641-646 (1997), forexample, report the enhancement of immune responses produced by DNAimmunizations when DNA sequences encoding molecules which stimulate theimmune response are co-administered. In a similar fashion, the vaccinesof the present invention may be co-administered with either nucleicacids encoding immune modulators or the immune modulators themselves.These immune modulators include granulocyte macrophage colonystimulating factor (GM-CSF) and CD86.

The vaccines of the present invention may be used to confer resistanceto borrelial infection by either passive or active immunization. Whenthe vaccines of the present invention are used to confer resistance toborrelial infection through active immunization, a vaccine of thepresent invention is administered to an animal to elicit a protectiveimmune response which either prevents or attenuates a borrelialinfection. When the vaccines of the present invention are used to conferresistance to borrelial infection through passive immunization, thevaccine is provided to a host animal (e.g., human, dog, or mouse), andthe antisera elicited by this antisera is recovered and directlyprovided to a recipient suspected of having an infection caused by amember of the Borrelia genus.

The ability to label antibodies, or fragments of antibodies, with toxinmolecules provides an additional method for treating borrelialinfections when passive immunization is conducted. In this embodiment,antibodies, or fragments of antibodies, capable of recognizing the B.burgdorferi polypeptides disclosed herein, or fragments thereof, as wellas other Borrelia proteins, are labeled with toxin molecules prior totheir administration to the patient. When such toxin derivatizedantibodies bind to Borrelia cells, toxin moieties will be localized tothese cells and will cause their death.

The present invention thus concerns and provides a means for preventingor attenuating a borrelial infection resulting from organisms which haveantigens that are recognized and bound by antisera produced in responseto the polypeptides of the present invention. As used herein, a vaccineis said to prevent or attenuate a disease if its administration to ananimal results either in the total or partial attenuation (i.e.,suppression) of a symptom or condition of the disease, or in the totalor partial immunity of the animal to the disease.

The administration of the vaccine (or the antisera which it elicits) maybe for either a “prophylactic” or “therapeutic” purpose. When providedprophylactically, the compound(s) are provided in advance of anysymptoms of borrelial infection. The prophylactic administration of thecompound(s) serves to prevent or attenuate any subsequent infection.When provided therapeutically, the compound(s) is provided upon or afterthe detection of symptoms which indicate that an animal may be infectedwith a member of the Borrelia genus. The therapeutic administration ofthe compound(s) serves to attenuate any actual infection. Thus, the B.burgdorferi polypeptides, and fragments thereof, of the presentinvention may be provided either prior to the onset of infection (so asto prevent or attenuate an anticipated infection) or after theinitiation of an actual infection.

The polypeptides of the invention, whether encoding a portion of anative protein or a functional derivative thereof, may be administeredin pure form or may be coupled to a macromolecular carrier. Example ofsuch carriers are proteins and carbohydrates. Suitable proteins whichmay act as macromolecular carrier for enhancing the immunogenicity ofthe polypeptides of the present invention include keyhole limpethemacyanin (KLH) tetanus toxoid, pertussis toxin, bovine serum albumin,and ovalbumin. Methods for coupling the polypeptides of the presentinvention to such macromolecular carriers are disclosed in Harlow etal., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988), the entire disclosureof which is incorporated by reference herein.

A composition is said to be “pharmacologically acceptable” if itsadministration can be tolerated by a recipient animal and is otherwisesuitable for administration to that animal. Such an agent is said to beadministered in a “therapeutically effective amount” if the amountadministered is physiologically significant. An agent is physiologicallysignificant if its presence results in a detectable change in thephysiology of a recipient patient.

While in all instances the vaccine of the present invention isadministered as a pharmacologically acceptable compound, one skilled inthe art would recognize that the composition of a pharmacologicallyacceptable compound varies with the animal to which it is administered.For example, a vaccine intended for human use will generally not beco-administered with Freund's adjuvant. Further, the level of purity ofthe B. burgdorferi polypeptides of the present invention will normallybe higher when administered to a human than when administered to anon-human animal.

As would be understood by one of ordinary skill in the art, when thevaccine of the present invention is provided to an animal, it may be ina composition which may contain salts, buffers, adjuvants, or othersubstances which are desirable for improving the efficacy of thecomposition. Adjuvants are substances that can be used to specificallyaugment a specific immune response. These substances generally performtwo functions: (1) they protect the antigen(s) from being rapidlycatabolized after administration and (2) they nonspecifically stimulateimmune responses.

Normally, the adjuvant and the composition are mixed prior topresentation to the immune system, or presented separately, but into thesame site of the animal being immunized. Adjuvants can be looselydivided into several groups based upon their composition. These groupsinclude oil adjuvants (for example, Freund's complete and incomplete),mineral salts (for example, AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄(SO₄), silica,kaolin, and carbon), polynucleotides (for example, poly IC and poly AUacids), and certain natural substances (for example, wax D fromMycobacterium tuberculosis, as well as substances found inCorynebacterium parvum, or Bordetella pertussis, and members of thegenus Brucella. Other substances useful as adjuvants are the saponinssuch as, for example, Quil A. (Superfos A/S, Denmark). Preferredadjuvants for use in the present invention include aluminum salts, suchas AlK(SO₄)₂, AlNa(SO₄)₂, and AlNH₄(SO₄). Examples of materials suitablefor use in vaccine compositions are provided in Remington'sPharmaceutical Sciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa.,pp. 1324-1341 (1980), which reference is incorporated herein byreference).

The therapeutic compositions of the present invention can beadministered parenterally by injection, rapid infusion, nasopharyngealabsorption (intranasopharangeally), dermoabsorption, or orally. Thecompositions may alternatively be administered intramuscularly, orintravenously. Compositions for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

Therapeutic compositions of the present invention can also beadministered in encapsulated form. For example, intranasal immunizationof mice against Bordetella pertussis infection using vaccinesencapsulated in biodegradable microsphere composed ofpoly(DL-lactide-co-glycolide) has been shown to stimulate protectiveimmune responses. Shahin, R. et al., Infect. Immun. 63: 1195-1200(1995). Similarly, orally administered encapsulated Salmonellatyphimurium antigens have also been shown to elicit protective immunityin mice. Allaoui-Attarki, K. et al., Infect. Immun. 65: 853-857 (1997).Encapsulated vaccines of the present invention can be administered by avariety of routes including those involving contacting the vaccine withmucous membranes (e.g., intranasally, intracolonicly, intraduodenally).

Many different techniques exist for the timing of the immunizations whena multiple administration regimen is utilized. It is possible to use thecompositions of the invention more than once to increase the levels anddiversities of expression of the immunoglobulin repertoire expressed bythe immunized animal. Typically, if multiple immunizations are given,they will be given one to two months apart.

According to the present invention, an “effective amount” of atherapeutic composition is one which is sufficient to achieve a desiredbiological effect. Generally, the dosage needed to provide an effectiveamount of the composition will vary depending upon such factors as theanimal's or human's age, condition, sex, and extent of disease, if any,and other variables which can be adjusted by one of ordinary skill inthe art.

The antigenic preparations of the invention can be administered byeither single or multiple dosages of an effective amount. Effectiveamounts of the compositions of the invention can vary from 0.01-1,000μg/ml per dose, more preferably 0.1-500 μg/ml per dose, and mostpreferably 10-300 μg/ml per dose.

Having now generally described the invention, the same will be morereadily understood through reference to the following example which isprovided by way of illustration, and is not intended to be limiting ofthe present invention, unless specified.

EXAMPLES

1. Preparation of PCR Primers and Amplification of DNA

Various fragments of the Borrelia burgdorferi genome, such as those ofTable 1, can be used, in accordance with the present invention, toprepare PCR primers for a variety of uses. The PCR primers arepreferably at least 15 bases, and more preferably at least 18 bases inlength. When selecting a primer sequence, it is preferred that theprimer pairs have approximately the same G/C ratio, so that meltingtemperatures are approximately the same. The PCR primers and amplifiedDNA of this Example find use in the Examples that follow.

2. Isolation of a Selected DNA Clone from B. burgdorferi

Three approaches are used to isolate a B. burgdorferi clone comprising apolynucleotide of the present invention from any B. burgdorferi genomicDNA library. The B. burgdorferi strain B31PU has been deposited as aconvienent source for obtaining a B. burgdorferi strain although a widevarity of strains B. burgdorferi strains can be used which are known inthe art.

B. burgdorferi genomic DNA is prepared using the following method. A 20ml overnight bacterial culture grown in a rich medium (e.g., TrypticaseSoy Broth, Brain Heart Infusion broth or Super broth), pelleted, ishedtwo times with TES (30 mM Tris-pH 8.0, 25 mM EDTA, 50 mM NaCl), andresuspended in 5 ml high salt TES (2.5M NaCl). Lysostaphin is added tofinal concentration of approx 50 ug/ml and the mixture is rotated slowly1 hour at 37 C to make protoplast cells. The solution is then placed inincubator (or place in a shaking water bath) and warmed to 55 C. Fivehundred micro liter of 20% sarcosyl in TES (final concentration 2%) isthen added to lyse the cells. Next, guanidine HCl is added to a finalconcentration of 7M (3.69 g in 5.5 ml). The mixture is swirled slowly at55 C for 60-90 min (solution should clear). A CsCl gradient is then setup in SW41 ultra clear tubes using 2.0 ml 5.7M CsCl and overlaying with2.85M CsCl. The gradient is carefully overlayed with the DNA-containingGuHCl solution. The gradient is spun at 30,000 rpm, 20 C for 24 hr andthe lower DNA band is collected. The volume is increased to 5 ml with TEbuffer. The DNA is then treated with protease K (10 ug/ml) overnight at37 C, and precipitated with ethanol. The precipitated DNA is resuspendedin a desired buffer.

In the first method, a plasmid is directly isolated by screening aplasmid B. burgdorferi genomic DNA library using a polynucleotide probecorresponding to a polynucleotide of the present invention.Particularly, a specific polynucleotide with 30-40 nucleotides issynthesized using an Applied Biosystems DNA synthesizer according to thesequence reported. The oligonucleotide is labeled, for instance, with³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (See, e.g., Maniatis et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).)The library is transformed into a suitable host, as indicated above(such as XL-1 Blue (Stratagene)) using techniques known to those ofskill in the art. See, e.g., Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel etal., CURRENT PROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y.1989). The transformants are plated on 1.5% agar plates (containing theappropriate selection agent, e.g., ampicillin) to a density of about 150transformants (colonies) per plate. These plates are screened usingNylon membranes according to routine methods for bacterial colonyscreening. See, e.g., Sambrook et al. MOLECULAR CLONING: A LABORATORYMANUAL (Cold Spring Harbor, N.Y. 2nd ed. 1989); Ausubel et al., CURRENTPROTOCALS IN MOLECULAR BIOLOGY (John Wiley and Sons, N.Y. 1989) or othertechniques known to those of skill in the art.

Alternatively, two primers of 15-25 nucleotides derived from the 5′ and3 ends of a polynucleotide of Table 1 are synthesized and used toamplify the desired DNA by PCR using a B. burgdorferi genomic DNA prepas a template. PCR is carried out under routine conditions, forinstance, in 25 VI of reaction mixture with 0.5 ug of the above DNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v)gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C.for 1 min) are performed with a Perkin-Elmer Cetus automated thermalcycler. The amplified product is analyzed by agarose gel electrophoresisand the DNA band with expected molecular weight is excised and purified.The PCR product is verified to be the selected sequence by subcloningand sequencing the DNA product.

Finally, overlapping oligos of the DNA sequences of Table 1 can bechemically synthesized and used to generate a nucleotide sequence ofdesired length using PCR methods known in the art.

3(a). Expression and Purification Borrelia Polypeptides in E. coli

The bacterial expression vector pQE60 is used for bacterial expressionof some of the polypeptide fragments of the present invention. (QIAGEN,Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311). pQE60 encodesampicillin antibiotic resistance (“Ampr”) and contains a bacterialorigin of replication (“ori”), an IPTG inducible promoter, a ribosomebinding site (“RBS”), six codons encoding histidine residues that allowaffinity purification using nickel-nitrilo-tri-acetic acid (“Ni-NTA”)affinity resin (QIAGEN, Inc., supra) and suitable single restrictionenzyme cleavage sites. These elements are arranged such that an insertedDNA fragment encoding a polypeptide expresses that polypeptide with thesix His residues (i.e., a “6×His tag”) covalently linked to the carboxylterminus of that polypeptide.

The DNA sequence encoding the desired portion of a B. burgdorferiprotein of the present invention is amplified from B. burgdorferigenomic DNA using PCR oligonucleotide primers which anneal to the 5′ and3′ sequences coding for the portions of the B. burgdorferipolynucleotide shown in Table 1. Additional nucleotides containingrestriction sites to facilitate cloning in the pQE60 vector are added tothe 5′ and 3′ sequences, respectively.

For cloning the mature protein, the 5′ primer has a sequence containingan appropriate restriction site followed by nucleotides of the aminoterminal coding sequence of the desired B. burgdorferi polynucleotidesequence in Table 1. One of ordinary skill in the art would appreciatethat the point in the protein coding sequence where the 5′ and 3′primers begin may be varied to amplify a DNA segment encoding anydesired portion of the complete protein shorter or longer than themature form. The 3′ primer has a sequence containing an appropriaterestriction site followed by nucleotides complementary to the 3′ end ofthe polypeptide coding sequence of Table 1, excluding a stop codon, withthe coding sequence aligned with the restriction site so as to maintainits reading frame with that of the six His codons in the pQE60 vector.

The amplified B. burgdorferi DNA fragment and the vector pQE60 aredigested with restriction enzymes which recognize the sites in theprimers and the digested DNAs are then ligated together. The B.burgdorferi DNA is inserted into the restricted pQE60 vector in a mannerwhich places the B. burgdorferi protein coding region downstream fromthe IPTG-inducible promoter and in-frame with an initiating AUG and thesix histidine codons.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described by Sambrook et al., supra.E. coli strain M15/rep4, containing multiple copies of the plasmidpREP4, which expresses the lac repressor and confers kanamycinresistance (“Kanr”), is used in carrying out the illustrative exampledescribed herein. This strain, which is only one of many that aresuitable for expressing a B. burgdorferi polypeptide, is availablecommercially (QIAGEN, Inc., supra). Transformants are identified bytheir ability to grow on LB agar plates in the presence of ampicillinand kanamycin. Plasmid DNA is isolated from resistant colonies and theidentity of the cloned DNA confirmed by restriction analysis, PCR andDNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-β-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C. in 6M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the B. burgdorferi polypeptide is loaded onto anickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin column (QIAGEN,Inc., supra). Proteins with a 6×His tag bind to the Ni-NTA resin withhigh affinity are purified in a simple one-step procedure (for detailssee: The QIAexpressionist, 1995, QIAGEN, Inc., supra). Briefly thesupernatant is loaded onto the column in 6 M guanidine-HCl, pH 8, thecolumn is first washed with 10 volumes of 6 M guanidine-HCl, pH 8, thenwashed with 10 volumes of 6 M guanidine-HCl pH 6, and finally the B.burgdorferi polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the protein could be successfully refoldedwhile immobilized on the Ni-NTA column. The recommended conditions areas follows: renature using a linear 6M-1M urea gradient in 500 mM NaCl,20% glycerol, 20 mM Tris/HCl pH 7.4, containing protease inhibitors. Therenaturation should be performed over a period of 1.5 hours or more.After renaturation the proteins can be eluted by the addition of 250 mMimmidazole. Immidazole is removed by a final dialyzing step against PBSor 50 mM sodium acetate pH 6 buffer plus 200 mM NaCl. The purifiedprotein is stored at 4° C. or frozen at −80° C.

The polypeptide of the present invention are also prepared using anon-denaturing protein purification method. For these polypeptides, thecell pellet from each liter of culture is resuspended in 25 mls of LysisBuffer A at 4° C. (Lysis Buffer A=50 mM Na-phosphate, 300 mM NaCl, 10 mM2-mercaptoethanol, 10% Glycerol, pH 7.5 with 1 tablet of CompleteEDTA-free protease inhibitor cocktail (Boehringer Mannheim #1873580) per50 ml of buffer). Absorbance at 550 nm is approximately 10-20 O.D./ml.The suspension is then put through three freeze/thaw cycles from −70° C.(using a ethanol-dry ice bath) up to room temperature. The cells arelysed via sonication in short 10 sec bursts over 3 minutes atapproximately 80 W while kept on ice. The sonicated sample is thencentrifuged at 15,000 RPM for 30 minutes at 4° C. The supernatant ispassed through a column containing 1.0 ml of CL-4B resin to pre-clearthe sample of any proteins that may bind to agarose non-specifically,and the flow-through fraction is collected.

The pre-cleared flow-through is applied to a nickel-nitrilo-tri-aceticacid (“Ni-NTA”) affinity resin column (Qiagen, Inc., supra). Proteinswith a 6×His tag bind to the Ni-NTA resin with high affinity and can bepurified in a simple one-step procedure. Briefly, the supernatant isloaded onto the column in Lysis Buffer A at 4° C., the column is firstwashed with 10 volumes of Lysis Buffer A until the A280 of the eluatereturns to the baseline. Then, the column is washed with 5 volumes of 40mM Imidazole (92% Lysis Buffer A/8% Buffer B) (Buffer B=50 mMNa-Phosphate, 300 mM NaCl, 10% Glycerol, 10 mM 2-mercaptoethanol, 500 mMImidazole, pH of the final buffer should be 7.5). The protein is elutedoff of the column with a series of increasing Imidazole solutions madeby adjusting the ratios of Lysis Buffer A to Buffer B. Three differentconcentrations are used: 3 volumes of 75 mM Imidazole, 3 volumes of 150mM Imidazole, 5 volumes of 500 mM Imidazole. The fractions containingthe purified protein are analyzed using 8%, 10% or 14% SDS-PAGEdepending on the protein size. The purified protein is then dialyzed 2×against phosphate-buffered saline (PBS) in order to place it into aneasily workable buffer. The purified protein is stored at 4° C. orfrozen at −80°.

The following alternative method may be used to purify B. burgdorferiexpressed in E coli when it is present in the form of inclusion bodies.Unless otherwise specified, all of the following steps are conducted at4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfluidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the B.burgdorferi polypeptide-containing supernatant is incubated at 4° C.overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded B. burgdorferi polypeptide solution, apreviously prepared tangential filtration unit equipped with 0.16 μmmembrane filter with appropriate surface area (e.g., Filtron),equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filteredsample is loaded onto a cation exchange resin (e.g., Poros HS-50,Perseptive Biosystems). The column is washed with 40 mM sodium acetate,pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in thesame buffer, in a stepwise manner. The absorbance at 280 mm of theeffluent is continuously monitored. Fractions are collected and furtheranalyzed by SDS-PAGE.

Fractions containing the B. burgdorferi polypeptide are then pooled andmixed with 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the B. burgdorferi polypeptide (determined, for instance, by16% SDS-PAGE) are then pooled.

The resultant B. burgdorferi polypeptide exhibits greater than 95%purity after the above refolding and purification steps. No majorcontaminant bands are observed from Commassie blue stained 16% SDS-PAGEgel when 5 μg of purified protein is loaded. The purified protein isalso tested for endotoxin/LPS contamination, and typically the LPScontent is less than 0.1 ng/ml according to LAL assays.

3(b). Alternative Expression and Purification Borrelia Polypeptides inE. coli

The vector pQE10 is alternatively used to clone and express some of thepolypeptides of the present invention for use in the soft tissue andsystemic infection models discussed below. The difference being suchthat an inserted DNA fragment encoding a polypeptide expresses thatpolypeptide with the six His residues (i.e., a “6×His tag”) covalentlylinked to the amino terminus of that polypeptide. The bacterialexpression vector pQE10 (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth,Calif., 91311) was used in this example. The components of the pQE10plasmid are arranged such that the inserted DNA sequence encoding apolypeptide of the present invention expresses the polypeptide with thesix His residues (i.e., a “6×His tag”)) covalently linked to the aminoterminus.

The DNA sequences encoding the desired portions of a polypeptide ofTable 1 were amplified using PCR oligonucleotide primers from genomic B.burgdorferi DNA. The PCR primers anneal to the nucleotide sequencesencoding the desired amino acid sequence of a polypeptide of the presentinvention. Additional nucleotides containing restriction sites tofacilitate cloning in the pQE10 vector were added to the 5′ and 3′primer sequences, respectively.

For cloning a polypeptide of the present invention, the 5′ and 3′primers were selected to amplify their respective nucleotide codingsequences. One of ordinary skill in the art would appreciate that thepoint in the protein coding sequence where the 5′ and 3′ primers beginsmay be varied to amplify a DNA segment encoding any desired portion of apolypeptide of the present invention. The 5′ primer was designed so thecoding sequence of the 6×His tag is aligned with the restriction site soas to maintain its reading frame with that of B. burgdorferipolypeptide. The 3′ was designed to include an stop codon. The amplifiedDNA fragment was then cloned, and the protein expressed, as describedabove for the pQE60 plasmid.

The DNA sequences of Table 1 encoding amino acid sequences may also becloned and expressed as fusion proteins by a protocol similar to thatdescribed directly above, wherein the pET-32b(+) vector (Novagen, 601Science Drive, Madison, Wis. 53711) is preferentially used in place ofpQE10.

The above methods are not limited to the polypeptide fragments actuallyproduced. The above method, like the methods below, can be used toproduce either full length polypeptides or desired fragments thereof.

3(c). Alternative Expression and Purification of Borrelia Polypeptidesin E. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). However, in this example, the polypeptide coding sequence isinserted such that translation of the six His codons is prevented and,therefore, the polypeptide is produced with no 6×His tag.

The DNA sequence encoding the desired portion of the B. burgdorferiamino acid sequence is amplified from an B. burgdorferi genomic DNA prepthe deposited DNA clones using PCR oligonucleotide primers which annealto the 5′ and 3′ nucleotide sequences corresponding to the desiredportion of the B. burgdorferi polypeptides. Additional nucleotidescontaining restriction sites to facilitate cloning in the pQE60 vectorare added to the 5′ and 3′ primer sequences.

For cloning a B. burgdorferi polypeptides of the present invention, 5′and 3′ primers are selected to amplify their respective nucleotidecoding sequences. One of ordinary skill in the art would appreciate thatthe point in the protein coding sequence where the 5′ and 3′ primersbegin may be varied to amplify a DNA segment encoding any desiredportion of a polypeptide of the present invention. The 3′ and 5′ primerscontain appropriate restriction sites followed by nucleotidescomplementary to the 5′ and 3′ ends of the coding sequence respectively.The 3′ primer is additionally designed to include an in-frame stopcodon.

The amplified B. burgdorferi DNA fragments and the vector pQE60 aredigested with restriction enzymes recognizing the sites in the primersand the digested DNAs are then ligated together. Insertion of the B.burgdorferi DNA into the restricted pQE60 vector places the B.burgdorferi protein coding region including its associated stop codondownstream from the IPTG-inducible promoter and in-frame with aninitiating AUG. The associated stop codon prevents translation of thesix histidine codons downstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described by Sambrook et al. E. colistrain M15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance (“Kanr”),is used in carrying out the illustrative example described herein. Thisstrain, which is only one of many that are suitable for expressing B.burgdorferi polypeptide, is available commercially (QIAGEN, Inc.,supra). Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

To purify the B. burgdorferi polypeptide, the cells are then stirred for3-4 hours at 4° C. in 6M guanidine-HCl, pH 8. The cell debris is removedby centrifugation, and the supernatant containing the B. burgdorferipolypeptide is dialyzed against 50 mM Na-acetate buffer pH 6,supplemented with 200 mM NaCl. Alternatively, the protein can besuccessfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol,25 mM Tris/HCl pH 7.4, containing protease inhibitors. Afterrenaturation the protein can be purified by ion exchange, hydrophobicinteraction and size exclusion chromatography. Alternatively, anaffinity chromatography step such as an antibody column can be used toobtain pure B. burgdorferi polypeptide. The purified protein is storedat 4° C. or frozen at −80° C.

The following alternative method may be used to purify B. burgdorferipolypeptides expressed in E coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells are harvested bycontinuous centrifugation at 15,000 rpm (Heraeus Sepatech). On the basisof the expected yield of protein per unit weight of cell paste and theamount of purified protein required, an appropriate amount of cellpaste, by weight, is suspended in a buffer solution containing 100 mMTris, 50 mM EDTA, pH 7.4. The cells are dispersed to a homogeneoussuspension using a high shear mixer.

The cells ware then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the B.burgdorferi polypeptide-containing supernatant is incubated at 4° C.overnight to allow further GuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded B. burgdorferi polypeptide solution, apreviously prepared tangential filtration unit equipped with 0.16 μmmembrane filter with appropriate surface area (e.g., Filtron),equilibrated with 40 mM sodium acetate, pH 6.0 is employed. The filteredsample is loaded onto a cation exchange resin (e.g., Poros HS-50,Perseptive Biosystems). The column is washed with 40 mM sodium acetate,pH 6.0 and eluted with 250 mM, 500 mM, 1000 mM, and 1500 mM NaCl in thesame buffer, in a stepwise manner. The absorbance at 280 mm of theeffluent is continuously monitored. Fractions are collected and furtheranalyzed by SDS-PAGE.

Fractions containing the B. burgdorferi polypeptide are then pooled andmixed with 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the B. burgdorferi polypeptide (determined, for instance, by16% SDS-PAGE) are then pooled.

The resultant B. burgdorferi polypeptide exhibits greater than 95%purity after the above refolding and purification steps. No majorcontaminant bands are observed from Commassie blue stained 16% SDS-PAGEgel when 5 μg of purified protein is loaded. The purified protein isalso tested for endotoxin/LPS contamination, and typically the LPScontent is less than 0.1 ng/ml according to LAL assays.

3(d). Cloning and Expression of B. burgdorferi in Other Bacteria

B. burgdorferi polypeptides can also be produced in: B. burgdorferiusing the methods of S. Skinner et al., (1988) Mol. Microbiol. 2:289-297 or J. I. Moreno (1996) Protein Expr. Purif. 8(3): 332-340;Lactobacillus using the methods of C. Rush et al., 1997 Appl. Microbiol.Biotechnol. 47(5): 537-542; or in Bacillus subtilis using the methodsChang et al., U.S. Pat. No. 4,952,508.

Cloning and Expression in COS Cells

A B. burgdorferi expression plasmid is made by cloning a portion of theDNA encoding a B. burgdorferi polypeptide into the expression vectorpDNAI/Amp or pDNAIII (which can be obtained from Invitrogen, Inc.). Theexpression vector pDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a DNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al. 1984 Cell 37: 767. The fusion of the HA tag to the targetprotein allows easy detection and recovery of the recombinant proteinwith an antibody that recognizes the HA epitope. pDNAIII contains, inaddition, the selectable neomycin marker.

A DNA fragment encoding a B. burgdorferi polypeptide is cloned into thepolylinker region of the vector so that recombinant protein expressionis directed by the CMV promoter. The plasmid construction strategy is asfollows. The DNA from a B. burgdorferi genomic DNA prep is amplifiedusing primers that contain convenient restriction sites, much asdescribed above for construction of vectors for expression of B.burgdorferi in E. coli. The 5′ primer contains a Kozak sequence, an AUGstart codon, and nucleotides of the 5′ coding region of the B.burgdorferi polypeptide. The 3′ primer, contains nucleotidescomplementary to the 3′ coding sequence of the B. burgdorferi DNA, astop codon, and a convenient restriction site.

The PCR amplified DNA fragment and the vector, pDNAI/Amp, are digestedwith appropriate restriction enzymes and then ligated. The ligationmixture is transformed into an appropriate E. coli strain such as SURE™(Stratagene Cloning Systems, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the fragment encoding the B.burgdorferi polypeptide.

For expression of a recombinant B. burgdorferi polypeptide, COS cellsare transfected with an expression vector, as described above, usingDEAE-dextran, as described, for instance, by Sambrook et al. (supra).Cells are incubated under conditions for expression of B. burgdorferi bythe vector.

Expression of the B. burgdorferi-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample Harlow et al., supra. To this end, two days after transfection,the cells are labeled by incubation in media containing ³⁵S-cysteine for8 hours. The cells and the media are collected, and the cells are washedand the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1%NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described byWilson et al. (supra). Proteins are precipitated from the cell lysateand from the culture media using an HA-specific monoclonal antibody. Theprecipitated proteins then are analyzed by SDS-PAGE and autoradiography.An expression product of the expected size is seen in the cell lysate,which is not seen in negative controls.

5. Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of B. burgdorferi polypeptidein this example. Plasmid pC4 is a derivative of the plasmid pSV2-dhfr(ATCC Accession No. 37146). The plasmid contains the mouse DHFR geneunder control of the SV40 early promoter. Chinese hamster ovary cells orother cells lacking dihydrofolate activity that are transfected withthese plasmids can be selected by growing the cells in a selectivemedium (alpha minus MEM, Life Technologies) supplemented with thechemotherapeutic agent methotrexate. The amplification of the DHFR genesin cells resistant to methotrexate (MTX) has been well documented. See,e.g., Alt et al., 1978, J. Biol. Chem. 253: 1357-1370; Hamlin et al.,1990, Biochem. et Biophys. Acta, 1097: 107-143; Page et al., 1991,Biotechnology 9: 64-68. Cells grown in increasing concentrations of MTXdevelop resistance to the drug by overproducing the target enzyme, DHFR,as a result of amplification of the DHFR gene. If a second gene islinked to the DHFR gene, it is usually co-amplified and over-expressed.It is known in the art that this approach may be used to develop celllines carrying more than 1,000 copies of the amplified gene(s).Subsequently, when the methotrexate is withdrawn, cell lines areobtained which contain the amplified gene integrated into one or morechromosome(s) of the host cell.

Plasmid pC4 contains the strong promoter of the long terminal repeat(LTR) of the Rouse Sarcoma Virus, for expressing a polypeptide ofinterest, Cullen, et al. (1985) Mol. Cell. Biol. 5: 438-447; plus afragment isolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV), Boshart, et al., 1985, Cell 41: 521-530.Downstream of the promoter are the following single restriction enzymecleavage sites that allow the integration of the genes: Bam HI, Xba I,and Asp 718. Behind these cloning sites the plasmid contains the 3′intron and polyadenylation site of the rat preproinsulin gene. Otherhigh efficiency promoters can also be used for the expression, e.g., thehuman β-actin promoter, the SV40 early or late promoters or the longterminal repeats from other retroviruses, e.g., HIV and HTLVI.Clontech's Tet-Off and Tet-On gene expression systems and similarsystems can be used to express the B. burgdorferi polypeptide in aregulated way in mammalian cells (Gossen et al., 1992, Proc. Natl. Acad.Sci. USA 89: 5547-5551. For the polyadenylation of the mRNA othersignals, e.g., from the human growth hormone or globin genes can be usedas well. Stable cell lines carrying a gene of interest integrated intothe chromosomes can also be selected upon co-transfection with aselectable marker such as gpt, G418 or hygromycin. It is advantageous touse more than one selectable marker in the beginning, e.g., G418 plusmethotrexate.

The plasmid pC4 is digested with the restriction enzymes and thendephosphorylated using calf intestinal phosphates by procedures known inthe art. The vector is then isolated from a 1% agarose gel. The DNAsequence encoding the B. burgdorferi polypeptide is amplified using PCRoligonucleotide primers corresponding to the 5′ and 3′ sequences of thedesired portion of the gene. A 5′ primer containing a restriction site,a Kozak sequence, an AUG start codon, and nucleotides of the 5′ codingregion of the B. burgdorferi polypeptide is synthesized and used. A 3′primer, containing a restriction site, stop codon, and nucleotidescomplementary to the 3′ coding sequence of the B. burgdorferipolypeptides is synthesized and used. The amplified fragment is digestedwith the restriction endonucleases and then purified again on a 1%agarose gel. The isolated fragment and the dephosphorylated vector arethen ligated with T4 DNA ligase. E. coli HB 101 or XL-1 Blue cells arethen transformed and bacteria are identified that contain the fragmentinserted into plasmid pC4 using, for instance, restriction enzymeanalysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. Five μg of the expression plasmid pC4 is cotransfectedwith 0.5 μg of the plasmid pSVneo using a lipid-mediated transfectionagent such as Lipofectin™ or LipofectAMINE™ (Life TechnologiesGaithersburg, Md.). The plasmid pSV2-neo contains a dominant selectablemarker, the neo gene from Tn5 encoding an enzyme that confers resistanceto a group of antibiotics including G418. The cells are seeded in alphaminus MEM supplemented with 1 mg/ml G418. After 2 days, the cells aretrypsinized and seeded in hybridoma cloning plates (Greiner, Germany) inalpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexateplus 1 mg/ml G418. After about 10-14 days single clones are trypsinizedand then seeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

6. Immunization and Detection of Immune Responses

6(a). B. burgdorferi Propagation

B. burgdorferi sensu stricto isolate B31 is propagated in tightly-closedcontainers at 34° C. in modified Barbour-Stoenner-Kelly (BSKII) medium(Barbour, A. G., Yale J. Biol. Med. 57: 521-525 (1984)) overlaid with a5% O₂/5% CO₂/90% N₂ gas mixture. Cell densities of these cultures aredetermined by darkfield microscopy at 400×.

Immunization of Mice and Challenge with B. burgdorferi. For activeimmunizations BALB/cByJ mice (BALB, Jackson Laboratories) are injectedintraperitoneally (i.p.) at week 0 with 20 g of recombinant borrelialprotein, or phosphate-buffered saline (PBS), emulsified with completeFreund's adjuvant (CFA), given a similar booster immunization inincomplete Freund's adjuvant (IFA) at week 4, and challenged at week 6.For challenge B. burgdorferi are diluted in BSKII fromexponentially-growing cultures and mice are injected subcutaneously(s.c.) at the base of the tail with 0.1 ml of these dilutions (typically10³-10⁴ borreliae; approximately 10-100 times the median infectiousdose). Borreliae used for challenge are passaged fewer than six times invitro. To assess infection, mice are sacrificed at 14-17 dayspost-challenge, and specimens derived from ear, bladder, and tibiotarsaljoints are placed in BSKII plus 1.4% gelatin, 13 g/ml amphotericin B,1.5 g/ml phosphomycin, and 15 g/ml rifampicin, and borrelia outgrowth attwo or three weeks is quantified by darkfield microscopy. Batches ofBSKII are qualified for infection testing by confirming that theysupported the growth of 1-5 cells of isolate B31. In some instancesseroconversion for protein P39 reactivity is also used to confirminfections (see below). Others have previously shown that mice elicitedantibodies to P39 when inoculated with live borreliae by syringe or tickbite, but not with killed borreliae (Simpson, W. J., et al., J. Clin.Microbiol. 29: 236-243 (1991)).

6(b). Immunoassays

Several immunoassay formats are used to quantify levels ofborrelia-specific antibodies (ELISA and immunoblot), and to evaluate thefunctional properties of these antibodies (growth inhibition assay). TheELISA and immunoblot assays are also used to detect and quantifyantibodies elicited in response to borrelial infection that react withspecific borrelial antigens. Where antibodies to certain borrelialantigens are elicited by infection this is taken as evidence that theborrelial proteins in question are expressed in vivo. Absence ofinfection-derived antibodies (seroconversion) following borrelialchallenge is evidence that infection is prevented or suppressed. Theimmunoblot assay is also used to ascertain whether antibodies raisedagainst recombinant borrelial antigens recognize a protein of similarsize in extracts of whole borreliae. Where the natural protein is ofsimilar, or identical, size in the immunoblot assay to the recombinantversion of the same protein, this is taken as evidence that therecombinant protein is the product of a full-length clone of therespective gene.

Enzyme-Linked Immunosorbant Assay (ELISA). The ELISA is used to quantifylevels of antibodies reactive with borrelial antigens elicited inresponse to immunization with these borrelial antigens. Wells of 96 wellmicrotiter plates (Immunlon 4, Dynatech, Chantilly, Va., or equivalent)are coated with antigen by incubating 50 l of 1 g/ml protein antigensolution in a suitable buffer, typically 0.1 M sodium carbonate bufferat pH 9.6. After decanting unbound antigen, additional binding sites areblocked by incubating 100 l of 3% nonfat milk in wash buffer (PBS, 0.2%Tween 20, pH 7.4). After washing, duplicate serial two-fold dilutions ofsera in PBS, Tween 20, 1% fetal bovine serum, are incubated for 1 hr,removed, wells are washed three times, and incubated with horseradishperoxidase-conjugated goat anti-mouse IgG. After three washes, boundantibodies are detected with H₂O₂ and2,2′-azino-di-(3-ethylbenzthiazoline sulfonate) (Schwan, T. G., et al.,Proc. Natl. Acad. Sci. USA 92: 2909-2913 (1985)) (ABTS®, Kirkegaard &Perry Labs., Gaithersburg, Md.) and A₄₀₅ is quantified with a MolecularDevices, Corp. (Menlo Park, Calif.) Vmax™ plate reader. IgG levels twicethe background level in serum from naive mice are assigned the minimumtiter of 1:100.

6(c). In Vitro Growth Inhibition Assay

Unlike other bacteria, borreliae can be killed by the binding ofspecific antibodies to their surface antigens. The mechanism for this invitro killing or growth-inhibitory effect is not known, but can occur inthe absence of serum complement, or other immune effector functions.Antibodies elicited in animals receiving immunizations with specificborrelial antigens that result in protection from borrelial challengeusually will directly kill borreliae in vitro. Thus, the in vitro growthinhibition assay also has a high predictive value for the protectivepotency of the borrelial antibodies, although exceptions, such asantibodies against OspC which are weak at in vitro growth inhibition,have been observed. Also, this assay can be used to evaluate theserologic conservation of epitope binding protective antibodies. Amicrowell antibody titration assay (Sadziene, A., et al., J. Infect.Dis. 167: 165-172 (1993)) is used to evaluate the growth inhibition (GI)properties of antisera against recombinant borrelial antigens againstthe homologous B31 isolate, and against various strains of borrelia.Briefly, 10⁵ borrelia in 100 l BSKII are added to serial two-folddilutions of sera in 100 l BSKII in 96-well plates, and the plates arecovered and incubated at 34° C. in a 5% O₂/5% CO₂/90% N₂ gas mixture for72 h prior to quantification of borrelia growth by darkfield microscopy.

6(d). Sodiumdodecylsulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)and Immunoblotting

Using a single well format, total borrelial protein extracts,recombinant borrelial antigen, or recombinant P39 samples (2 g ofpurified protein, or more for total borrelial extracts) are boiled inSDS/2-ME sample buffer before electrophoresis through 3% acrylamidestacking gels, and resolving gels of higher acrylamide concentration,typically 10-15% acrylamide monomer. Gels are electro-blotted tonitrocellulose membranes and lanes are probed with dilutions of antibodyto be tested for reactivity with specific borrelial antigens, followedby the appropriate secondary antibody-enzyme (horseradish peroxidase)conjugate. When it is desirable to confirm that the protein hadtransferred following electro-blotting, membranes are stained withPonceau S. Immunoblot signals from bound antibodies are detected onx-ray film as chemiluminescence using ECL™ reagents (Amersham Corp.,Arlington Heights, Ill.).

6(e). Detection of Borrelia mRNA Expression

Northern blot analysis is carried out using methods described by, amongothers, Sambrook et al., supra. to detect the expression of the B.burgdorferi nucleotide sequences of the present invention in animaltissues. A cDNA probe containing an entire nucleotide sequence shown inTable 1 is labeled with ³²P using the rediprime™ DNA labeling system(Amersham Life Science), according to manufacturer's instructions. Afterlabeling, the probe is purified using a CHROMA SPIN-100™ column(Clontech Laboratories, Inc.), according to manufacturer's protocolnumber PT1200-1. The purified labeled probe is then used to detect theexpression of Borrelia mRNA in an animal tissue sample.

Animal tissues, such as blood or spinal fluid, are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70 C overnight, and films developed according to standard procedures.

The disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference in theirentireties.

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention. Functionally equivalent methodsand components are within the scope of the invention, in addition tothose shown and described herein and will become apparent to thoseskilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims. TABLE 1 Nucleotide and Amino Acid Sequences.f101.aa (SEQ ID NO:1) t101.aa (SEQ ID NO:2) f101.nt (SEQ ID NO:3)t101.nt (SEQ ID NO:4) f11.aa (SEQ ID NO:5) t11.aa (SEQ ID NO:6) f11.nt(SEQ ID NO:7) t11.nt (SEQ ID NO:8) f12.aa (SEQ ID NO:9) t12.aa (SEQ IDNO:10) f12.nt (SEQ ID NO:11) t12.nt (SEQ ID NO:12) f129.aa (SEQ IDNO:13) t129.aa (SEQ ID NO:14) f129.nt (SEQ ID NO:15) t129.nt (SEQ IDNO:16) f142.aa (SEQ ID NO:17) t142.aa (SEQ ID NO:18) f142.nt (SEQ IDNO:19) t142.nt (SEQ ID NO:20) f147.aa (SEQ ID NO:21) t147.aa (SEQ IDNO:22) f147.nt (SEQ ID NO:23) t147.nt (SEQ ID NO:24) f152.aa (SEQ IDNO:25) t152.aa (SEQ ID NO:26) f152.nt (SEQ ID NO:27) t152.nt (SEQ IDNO:28) f154.aa (SEQ ID NO:29) t154.aa (SEQ ID NO:30) f154.nt (SEQ IDNO:31) t154.nt (SEQ ID NO:32) f157.aa (SEQ ID NO:33) t157.aa (SEQ IDNO:34) f157.nt (SEQ ID NO:35) t157.nt (SEQ ID NO:36) f17.aa (SEQ IDNO:37) t17.aa (SEQ ID NO:38) f17.nt (SEQ ID NO:39) t17.nt (SEQ ID NO:40)f170.aa (SEQ ID NO:41) t170.aa (SEQ ID NO:42) f170.nt (SEQ ID NO:43)t170.nt (SEQ ID NO:44) f186.aa (SEQ ID NO:45) t186.aa (SEQ ID NO:46)f186.nt (SEQ ID NO:47) t186.nt (SEQ ID NO:48) f196.aa (SEQ ID NO:49)t196.aa (SEQ ID NO:50) f196.nt (SEQ ID NO:51) t196.nt (SEQ ID NO:52)f899.aa (SEQ ID NO:53) t899.aa (SEQ ID NO:54) f899.nt (SEQ ID NO:55)t899.nt (SEQ ID NO:56) f924.aa (SEQ ID NO:57) t924.aa (SEQ ID NO:58)f924.nt (SEQ ID NO:59) t924.nt (SEQ ID NO:60) f925.aa (SEQ ID NO:61)t925.aa (SEQ ID NO:62) f925.nt (SEQ ID NO:63) t925.nt (SEQ ID NO:64)f929.aa (SEQ ID NO:65) t929.aa (SEQ ID NO:66) f929.nt (SEQ ID NO:67)t929.nt (SEQ ID NO:68) f933.aa (SEQ ID NO:69) t933.aa (SEQ ID NO:70)f933.nt (SEQ ID NO:71) t933.nt (SEQ ID NO:72) f940.aa (SEQ ID NO:73)t940.aa (SEQ ID NO:74) f940.nt (SEQ ID NO:75) t940.nt (SEQ ID NO:76)f943.aa (SEQ ID NO:77) t943.aa (SEQ ID NO:78) f943.nt (SEQ ID NO:79)t943.nt (SEQ ID NO:80) f952.aa (SEQ ID NO:81) t952.aa (SEQ ID NO:82)f952.nt (SEQ ID NO:83) t952.nt (SEQ ID NO:84) f378.aa (SEQ ID NO:85)t378.aa (SEQ ID NO:86) f378.nt (SEQ ID NO:87) t378.nt (SEQ ID NO:88)f4.aa (SEQ ID NO:89) t4.aa (SEQ ID NO:90) f4.nt (SEQ ID NO:91) t4.nt(SEQ ID NO:92) f43.aa (SEQ ID NO:93) t43.aa (SEQ ID NO:94) f43.nt (SEQID NO:95) t43.nt (SEQ ID NO:96) f50.aa (SEQ ID NO:97) t50.aa (SEQ IDNO:98) f50.nt (SEQ ID NO:99) t50.nt (SEQ ID NO:100) f65.aa (SEQ IDNO:101) t65.aa (SEQ ID NO:102) f65.nt (SEQ ID NO:103) t65.nt (SEQ IDNO:104) f8.aa (SEQ ID NO:105) t8.aa (SEQ ID NO:106) f8.nt (SEQ IDNO:107) t8.nt (SEQ ID NO:108) f82.aa (SEQ ID NO:109) t82.aa (SEQ IDNO:110) f82.nt (SEQ ID NO:111) f82.nt (SEQ ID NO:112) f86.aa (SEQ IDNO:113) t86.aa (SEQ ID NO:114) f86.nt (SEQ ID NO:115) t86.nt (SEQ IDNO:116) f90.aa (SEQ ID NO:117) t90.aa (SEQ ID NO:118) f90.nt (SEQ IDNO:119) t90.nt (SEQ ID NO:120) f469.aa (SEQ ID NO:121) t469.aa (SEQ IDNO:122) f469.nt (SEQ ID NO:123) t469.nt (SEQ ID NO:124) f477.aa (SEQ IDNO:125) t477.aa (SEQ ID NO:126) f477.nt (SEQ ID NO:127) t477.nt (SEQ IDNO:128) f488.aa (SEQ ID NO:129) t488.aa (SEQ ID NO:130) f488.nt (SEQ IDNO:131) t488.nt (SEQ ID NO:132) f494.aa (SEQ ID NO:133) t494.aa (SEQ IDNO:134) f494.nt (SEQ ID NO:135) t494.nt (SEQ ID NO:136) f516.aa (SEQ IDNO:137) t516.aa (SEQ ID NO:138) f516.nt (SEQ ID NO:139) t516.nt (SEQ IDNO:140) f517.aa (SEQ ID NO:141) t517.aa (SEQ ID NO:142) f517.nt (SEQ IDNO:143) t517.nt (SEQ ID NO:144) f519.aa (SEQ ID NO:145) t519.aa (SEQ IDNO:146) f519.nt (SEQ ID NO:147) t519.nt (SEQ ID NO:148) f520.aa (SEQ IDNO:149) t520.aa (SEQ ID NO:150) f520.nt (SEQ ID NO:151) t520.nt (SEQ IDNO:152) f523.aa (SEQ ID NO:153) t523.aa (SEQ ID NO:154) f523.nt (SEQ IDNO:155) f523.nt (SEQ ID NO:156) f526.aa (SEQ ID NO:157) t526.aa (SEQ IDNO:158) f526.nt (SEQ ID NO:159) t526.nt (SEQ ID NO:160) f544.aa (SEQ IDNO:161) t544.aa (SEQ ID NO:162) f544.nt (SEQ ID NO:163) t544.nt (SEQ IDNO:164) f545.aa (SEQ ID NO:165) t545.aa (SEQ ID NO:166) f545.nt (SEQ IDNO:167) t545.nt (SEQ ID NO:168) f577.aa (SEQ ID NO:169) t577.aa (SEQ IDNO:170) f577.nt (SEQ ID NO:171) t577.nt (SEQ ID NO:172) f584.aa (SEQ IDNO:173) t584.aa (SEQ ID NO:174) f584.nt (SEQ ID NO:175) t584.nt (SEQ IDNO:176) f596.aa (SEQ ID NO:177) t596.aa (SEQ ID NO:178) f596.nt (SEQ IDNO:179) t596.nt (SEQ ID NO:180) f598.aa (SEQ ID NO:181) t598.aa (SEQ IDNO:182) f598.nt (SEQ ID NO:183) t598.nt (SEQ ID NO:184) f600.aa (SEQ IDNO:185) t600.aa (SEQ ID NO:186) f600.nt (SEQ ID NO:187) t600.nt (SEQ IDNO:188) f603.aa (SEQ ID NO:189) t603.aa (SEQ ID NO:190) f603.nt (SEQ IDNO:191) t603.nt (SEQ ID NO:192) f607.aa (SEQ ID NO:193) t607.aa (SEQ IDNO:194) f607.nt (SEQ ID NO:195) t607.nt (SEQ ID NO:196) f611.aa (SEQ IDNO:197) t611.aa (SEQ ID NO:198) f611.nt (SEQ ID NO:199) t611.nt (SEQ IDNO:200) f617.aa (SEQ ID NO:201) t617.aa (SEQ ID NO:202) f617.nt (SEQ IDNO:203) t617.nt (SEQ ID NO:204) f631.aa (SEQ ID NO:205) t631.aa (SEQ IDNO:206) f631.nt (SEQ ID NO:207) t631.nt (SEQ ID NO:208) f647.aa (SEQ IDNO:209) t647.aa (SEQ ID NO:210) f647.nt (SEQ ID NO:211) t647.nt (SEQ IDNO:212) f653.aa (SEQ ID NO:213) t653.aa (SEQ ID NO:214) f653.nt (SEQ IDNO:215) t653.nt (SEQ ID NO:216) f664.aa (SEQ ID NO:217) t664.aa (SEQ IDNO:218) f664.nt (SEQ ID NO:219) t664.nt (SEQ ID NO:220) f680.aa (SEQ IDNO:221) t680.aa (SEQ ID NO:222) f680.nt (SEQ ID NO:223) t680.nt (SEQ IDNO:224) f688.aa (SEQ ID NO:225) t688.aa (SEQ ID NO:226) f688.nt (SEQ IDNO:227) t688.nt (SEQ ID NO:228) f704.aa (SEQ ID NO:229) t704.aa (SEQ IDNO:230) f704.nt (SEQ ID NO:231) t704.nt (SEQ ID NO:232) f707.aa (SEQ IDNO:233) t707.aa (SEQ ID NO:234) f707.nt (SEQ ID NO:235) t707.nt (SEQ IDNO:236) f709.aa (SEQ ID NO:237) t709.aa (SEQ ID NO:238) f709.nt (SEQ IDNO:239) t709.nt (SEQ ID NO:240) f730.aa (SEQ ID NO:241) t730.aa (SEQ IDNO:242) f730.nt (SEQ ID NO:243) t730.nt (SEQ ID NO:244) f197.aa (SEQ IDNO:245) t197.aa (SEQ ID NO:246) f197.nt (SEQ ID NO:247) t197.nt (SEQ IDNO:248) f200.aa (SEQ ID NO:249) t200.aa (SEQ ID NO:250) f200.nt (SEQ IDNO:251) t200.nt (SEQ ID NO:252) f208.aa (SEQ ID NO:253) t208.aa (SEQ IDNO:254) f208.nt (SEQ ID NO:255) t208.nt (SEQ ID NO:256) f210.aa (SEQ IDNO:257) t210.aa (SEQ ID NO:258) f210.nt (SEQ ID NO:259) t210.nt (SEQ IDNO:260) f22.aa (SEQ ID NO:261) t22.aa (SEQ ID NO:262) f22.nt (SEQ IDNO:263) t22.nt (SEQ ID NO:264) f221.aa (SEQ ID NO:265) t221.aa (SEQ IDNO:266) f221.nt (SEQ ID NO:267) t221.nt (SEQ ID NO:268) f253.aa (SEQ IDNO:269) t253.aa (SEQ ID NO:270) f253.nt (SEQ ID NO:271) t253.nt (SEQ IDNO:272) f265.aa (SEQ ID NO:273) t265.aa (SEQ ID NO:274) f265.nt (SEQ IDNO:275) t265.nt (SEQ ID NO:276) f269.aa (SEQ ID NO:277) t269.aa (SEQ IDNO:278) f269.nt (SEQ ID NO:279) t269.nt (SEQ ID NO:280) f29.aa (SEQ IDNO:281) t29.aa (SEQ ID NO:282) f29.nt (SEQ ID NO:283) t29.nt (SEQ IDNO:284) f290.aa (SEQ ID NO:285) t290.aa (SEQ ID NO:286) f290.nt (SEQ IDNO:287) t290.nt (SEQ ID NO:288) f291.aa (SEQ ID NO:289) t291.aa (SEQ IDNO:290) f291.nt (SEQ ID NO:291) t291.nt (SEQ ID NO:292) f296.aa (SEQ IDNO:293) t296.aa (SEQ ID NO:294) f296.nt (SEQ ID NO:295) t296.nt (SEQ IDNO:296) f3.aa (SEQ ID NO:297) t3.aa (SEQ ID NO:298) f3.nt (SEQ IDNO:299) t3.nt (SEQ ID NO:300) f30.aa (SEQ ID NO:301) t30.aa (SEQ IDNO:302) f30.nt (SEQ ID NO:303) t30.nt (SEQ ID NO:304) f308.aa (SEQ IDNO:305) t308.aa (SEQ ID NO:306) f308.nt (SEQ ID NO:307) t308.nt (SEQ IDNO:308) f31.aa (SEQ ID NO:309) t31.aa (SEQ ID NO:310) f31.nt (SEQ IDNO:311) t31.nt (SEQ ID NO:312) f939.aa (SEQ ID NO:313) f939.aa (SEQ IDNO:314) f939.nt (SEQ ID NO:315) t939.nt (SEQ ID NO:316) f739.aa (SEQ IDNO:317) t739.aa (SEQ ID NO:318) f739.nt (SEQ ID NO:319) t739.nt (SEQ IDNO:320) f742.aa (SEQ ID NO:321) t742.aa (SEQ ID NO:322) f742.nt (SEQ IDNO:323) t742.nt (SEQ ID NO:324) f743.aa (SEQ ID NO:325) t743.aa (SEQ IDNO:326) f743.nt (SEQ ID NO:327) t743.nt (SEQ ID NO:328) f748.aa (SEQ IDNO:329) t748.aa (SEQ ID NO:330) t748.nt (SEQ ID NO:331) t748.nt (SEQ IDNO:332) t764.aa (SEQ ID NO:333) f764.aa (SEQ ID NO:334) f764.nt (SEQ IDNO:335) t764.nt (SEQ ID NO:336) f770.aa (SEQ ID NO:337) t770.aa (SEQ IDNO:338) f770.nt (SEQ ID NO:339) t770.nt (SEQ ID NO:340) f790.aa (SEQ IDNO:341) t790.aa (SEQ ID NO:342) f790.nt (SEQ ID NO:343) t790.nt (SEQ IDNO:344) f792.aa (SEQ ID NO:345) t792.aa (SEQ ID NO:346) f792.nt (SEQ IDNO:347) t792.nt (SEQ ID NO:348) f797.aa (SEQ ID NO:349) t797.aa (SEQ IDNO:350) f797.nt (SEQ ID NO:351) t797.nt (SEQ ID NO:352) f799.aa (SEQ IDNO:353) t799.aa (SEQ ID NO:354) f799.nt (SEQ ID NO:355) t799.nt (SEQ IDNO:356) f800.aa (SEQ ID NO:357) t800.aa (SEQ ID NO:358) f800.nt (SEQ IDNO:359) t800.nt (SEQ ID NO:360) f810.aa (SEQ ID NO:361) t810.aa (SEQ IDNO:362) f810.nt (SEQ ID NO:363) t810.nt (SEQ ID NO:364) f814.aa (SEQ IDNO:365) t814.aa (SEQ ID NO:366) f814.nt (SEQ ID NO:367) t814.nt (SEQ IDNO:368) f818.aa (SEQ ID NO:369) t818.aa (SEQ ID NO:370) f818.nt (SEQ IDNO:371) t818.nt (SEQ ID NO:372) f820.aa (SEQ ID NO:373) t820.aa (SEQ IDNO:374) f820.nt (SEQ ID NO:375) t820.nt (SEQ ID NO:376) f831.aa (SEQ IDNO:377) t831.aa (SEQ ID NO:378) f831.nt (SEQ ID NO:379) t831.nt (SEQ IDNO:380) f843.aa (SEQ ID NO:381) t843.aa (SEQ ID NO:382) f843.nt (SEQ IDNO:383) t843.nt (SEQ ID NO:384) f850.aa (SEQ ID NO:385) t850.aa (SEQ IDNO:386) f850.nt (SEQ ID NO:387) t850.nt (SEQ ID NO:388) f853.aa (SEQ IDNO:389) t853.aa (SEQ ID NO:390) f853.nt (SEQ ID NO:391) t853.nt (SEQ IDNO:392) f859.aa (SEQ ID NO:393) t859.aa (SEQ ID NO:394) f859.nt (SEQ IDNO:395) t859.nt (SEQ ID NO:396) f861.aa (SEQ ID NO:397) t861.aa (SEQ IDNO:398) f861.nt (SEQ ID NO:399) t861.nt (SEQ ID NO:400) f363.aa (SEQ IDNO:401) t363.aa (SEQ ID NO:402) f363.nt (SEQ ID NO:403) t363.nt (SEQ IDNO:404) f368.aa (SEQ ID NO:405) t368.aa (SEQ ID NO:406) f368.nt (SEQ IDNO:407) t368.nt (SEQ ID NO:408) f371.aa (SEQ ID NO:409) t371.aa (SEQ IDNO:410) f371.nt (SEQ ID NO:411) t371.nt (SEQ ID NO:412) f502.aa (SEQ IDNO:413) t502.aa (SEQ ID NO:414) f502.nt (SEQ ID NO:415) t502.nt (SEQ IDNO:416) f527.aa (SEQ ID NO:417) t527.aa (SEQ ID NO:418) f527.nt (SEQ IDNO:419) t527.nt (SEQ ID NO:420) f541.aa (SEQ ID NO:421) t541.aa (SEQ IDNO:422) f541.nt (SEQ ID NO:423) t541.nt (SEQ ID NO:424) f561.aa (SEQ IDNO:425) t561.aa (SEQ ID NO:426) f561.nt (SEQ ID NO:427) t561.nt (SEQ IDNO:428) f604.aa (SEQ ID NO:429) t604.aa (SEQ ID NO:430) f604.nt (SEQ IDNO:431) t604.nt (SEQ ID NO:432) f736.aa (SEQ ID NO:433) t736.aa (SEQ IDNO:434) f736.nt (SEQ ID NO:435) t736.nt (SEQ ID NO:436) f752.aa (SEQ IDNO:437) t752.aa (SEQ ID NO:438) f752.nt (SEQ ID NO:439) t752.nt (SEQ IDNO:440) f798.aa (SEQ ID NO:441) t798.aa (SEQ ID NO:442) t798.nt (SEQ IDNO:443) t798.nt (SEQ ID NO:444) f805.aa (SEQ ID NO:445) t805.aa (SEQ IDNO:446) f805.nt (SEQ ID NO:447) t805.nt (SEQ ID NO:448) f635.aa (SEQ IDNO:449) t635.aa (SEQ ID NO:450) f635.nt (SEQ ID NO:451) t635.nt (SEQ IDNO:452) f314.aa (SEQ ID NO:453) t314.aa (SEQ ID NO:454) f314.nt (SEQ IDNO:455) t314.nt (SEQ ID NO:456) f32.aa (SEQ ID NO:457) t32.aa (SEQ IDNO:458) f32.nt (SEQ ID NO:459) t32.nt (SEQ ID NO:460) f320.aa (SEQ IDNO:461) t320.aa (SEQ ID NO:462) f320.nt (SEQ ID NO:463) t320.nt (SEQ IDNO:464) f342.aa (SEQ ID NO:465) t342.aa (SEQ ID NO:466) f342.nt (SEQ IDNO:467) t342.nt (SEQ ID NO:468) f352.aa (SEQ ID NO:469) t352.aa (SEQ IDNO:470) t352.nt (SEQ ID NO:471) t352.nt (SEQ ID NO:472) f301.aa (SEQ IDNO:473) t301.aa (SEQ ID NO:474) f301.nt (SEQ ID NO:475) t301.nt (SEQ IDNO:476) f346.aa (SEQ ID NO:477) t346.aa (SEQ ID NO:478) f346.nt (SEQ IDNO:479) t346.nt (SEQ ID NO:480) f373.aa (SEQ ID NO:481) t373.aa (SEQ IDNO:482) f373.nt (SEQ ID NO:483) t373.nt (SEQ ID NO:484) f384.aa (SEQ IDNO:485) t384.aa (SEQ ID NO:486) f384.nt (SEQ ID NO:487) t384.nt (SEQ IDNO:488) f860.aa (SEQ ID NO:489) t860.aa (SEQ ID NO:490) f860.nt (SEQ IDNO:491) t860.nt (SEQ ID NO:492) f446.aa (SEQ ID NO:493) t446.aa (SEQ IDNO:494) f446.nt (SEQ ID NO:495) t446.nt (SEQ ID NO:496) f457.aa (SEQ IDNO:497) t457.aa (SEQ ID NO:498) f457.nt (SEQ ID NO:499) t457.nt (SEQ IDNO:500) f542.aa (SEQ ID NO:501) t542.aa (SEQ ID NO:502) f542.nt (SEQ IDNO:503) t542.nt (SEQ ID NO:504) f93.aa (SEQ ID NO:505) t93.aa (SEQ IDNO:506) f93.nt (SEQ ID NO:507) t93.nt (SEQ ID NO:508) f105.aa (SEQ IDNO:509) t105.aa (SEQ ID NO:510) f105.nt (SEQ ID NO:511) t105.nt (SEQ IDNO:512) f150.aa (SEQ ID NO:513) t150.aa (SEQ ID NO:514) f150.nt (SEQ IDNO:515) t150.nt (SEQ ID NO:516) f219.aa (SEQ ID NO:517) t219.aa (SEQ IDNO:518) f219.nt (SEQ ID NO:519) t219.nt (SEQ ID NO:520) f229.aa (SEQ IDNO:521) t229.aa (SEQ ID NO:522) f229.nt (SEQ ID NO:523) t229.nt (SEQ IDNO:524) f22.aa (SEQ ID NO:525) t22.aa (SEQ ID NO:526) f22.nt (SEQ IDNO:527) t22.nt (SEQ ID NO:528) f32.aa (SEQ ID NO:529) t32.aa (SEQ IDNO:530) f32.nt (SEQ ID NO:531) t32.nt (SEQ ID NO:532) f186.aa (SEQ IDNO:533) t186.aa (SEQ ID NO:534) f186.nt (SEQ ID NO:535) t186.nt (SEQ IDNO:536) f216.aa (SEQ ID NO:537) t216.aa (SEQ ID NO:538) f216.nt (SEQ IDNO:539) t216.nt (SEQ ID NO:540) f328.aa (SEQ ID NO:541) t328.aa (SEQ IDNO:542) f328.nt (SEQ ID NO:543) t328.nt (SEQ ID NO:544) t352.aa (SEQ IDNO:545) t352.aa (SEQ ID NO:546) f352.nt (SEQ ID NO:547) t352.nt (SEQ IDNO:548) f867.aa (SEQ ID NO:549) t867.aa (SEQ ID NO:550) f867.nt (SEQ IDNO:551) t867.nt (SEQ ID NO:552) f868.aa (SEQ ID NO:553) t868.aa (SEQ IDNO:554) f868.nt (SEQ ID NO:555) t868.nt (SEQ ID NO:556) f872.aa (SEQ IDNO:557) t872.aa (SEQ ID NO:558) f872.nt (SEQ ID NO:559) t872.nt (SEQ IDNO:560) f874.aa (SEQ ID NO:561) t874.aa (SEQ ID NO:562) f874.nt (SEQ IDNO:563) t874.nt (SEQ ID NO:564) f886.aa (SEQ ID NO:565) t886.aa (SEQ IDNO:566) f886.nt (SEQ ID NO:567) t886.nt (SEQ ID NO:568) f888.aa (SEQ IDNO:569) t888.aa (SEQ ID NO:570) f888.nt (SEQ ID NO:571) t888.nt (SEQ IDNO:572) f893.aa (SEQ ID NO:573) t893.aa (SEQ ID NO:574) f893.nt (SEQ IDNO:575) t893.nt (SEQ ID NO:576) f895.aa (SEQ ID NO:577) t895.aa (SEQ IDNO:578) f895.nt (SEQ ID NO:579) t895.nt (SEQ ID NO:580) f605.aa (SEQ IDNO:581) t605.aa (SEQ ID NO:582) f605.nt (SEQ ID NO:583) t605.nt (SEQ IDNO:584) f606.aa (SEQ ID NO:585) t606.aa (SEQ ID NO:586) f606.nt (SEQ IDNO:587) t606.nt (SEQ ID NO:588) f679.aa (SEQ ID NO:589) t679.aa (SEQ IDNO:590) f679.nt (SEQ ID NO:591) t679.nt (SEQ ID NO:592) f11-12.nt (SEQID NO:593) t11-12.nt (SEQ ID NO:594) f11-12.aa (SEQ ID NO:595) t11-12.aa(SEQ ID NO:596) f11-4.nt (SEQ ID NO:597) t11-4.nt (SEQ ID NO:598)f11-4.aa (SEQ ID NO:599) t11-4.aa (SEQ ID NO:600) f112-1.nt (SEQ IDNO:601) t112-1.nt (SEQ ID NO:602) f112-1.aa (SEQ ID NO:603) t112-1.aa(SEQ ID NO:604) f14-8.nt (SEQ ID NO:605) t14-8.nt (SEQ ID NO:606)f14-8.aa (SEQ ID NO:607) t14-8.aa (SEQ ID NO:608) f17-6.nt (SEQ IDNO:609) t17-6.nt (SEQ ID NO:610) f17-6.aa (SEQ ID NO:611) t17-6.aa (SEQID NO:612) f19-2.nt (SEQ ID NO:613) t19-2.nt (SEQ ID NO:614) f19-2.aa(SEQ ID NO:615) t19-2.aa (SEQ ID NO:616) f19-4.nt (SEQ ID NO:617)t19-4.nt (SEQ ID NO:618) f19-4.aa (SEQ ID NO:619) t19-4.aa (SEQ IDNO:620) f19-6.nt (SEQ ID NO:621) t19-6.nt (SEQ ID NO:622) f19-6.aa (SEQID NO:623) t19-6.aa (SEQ ID NO:624) f21-4.nt (SEQ ID NO:625) t21-4.nt(SEQ ID NO:626) f21-4.aa (SEQ ID NO:627) t21-4.aa (SEQ ID NO:628)f24-1.nt (SEQ ID NO:629) t24-1.nt (SEQ ID NO:630) f24-1.aa (SEQ IDNO:631) t24-1.aa (SEQ ID NO:632) f28-2.nt (SEQ ID NO:633) t28-2.nt (SEQID NO:634) f28-2.aa (SEQ ID NO:635) t28-2.aa (SEQ ID NO:636) f28-3.nt(SEQ ID NO:637) t28-3.nt (SEQ ID NO:638) f28-3.aa (SEQ ID NO:639)t28-3.aa (SEQ ID NO:640) f31-2.nt (SEQ ID NO:641) t31-2.nt (SEQ IDNO:642) f31-2.aa (SEQ ID NO:643) t31-2.aa (SEQ ID NO:644) f32-4.nt (SEQID NO:645) t32-4.nt (SEQ ID NO:646) f32-4.aa (SEQ ID NO:647) t32-4.aa(SEQ ID NO:648) f4-15.nt (SEQ ID NO:649) t4-15.nt (SEQ ID NO:650)f4-15.aa (SEQ ID NO:651) t4-15.aa (SEQ ID NO:652) f4-50.nt (SEQ IDNO:653) t4-50.nt (SEQ ID NO:654) f4-50.aa (SEQ ID NO:655) t4-50.aa (SEQID NO:656) f4-66.nt (SEQ ID NO:657) t4-66.nt (SEQ ID NO:658) f4-66.aa(SEQ ID NO:659) t4-66.aa (SEQ ID NO:660) f42-1.nt (SEQ ID NO:661)t42-1.nt (SEQ ID NO:662) f42-1.aa (SEQ ID NO:663) t42-1.aa (SEQ IDNO:664) f43-3.nt (SEQ ID NO:665) t43-3.nt (SEQ ID NO:666) f43-3.aa (SEQID NO:667) t43-3.aa (SEQ ID NO:668) f45-2.nt (SEQ ID NO:669) t45-2.nt(SEQ ID NO:670) f45-2.aa (SEQ ID NO:671) t45-2.aa (SEQ ID NO:672)f47-2.nt (SEQ ID NO:673) t47-2.nt (SEQ ID NO:674) f47-2.aa (SEQ IDNO:675) t47-2.aa (SEQ ID NO:676) f49-2.nt (SEQ ID NO:677) t49-2.nt (SEQID NO:678) f49-2.aa (SEQ ID NO:679) t49-2.aa (SEQ ID NO:680) f5-14.nt(SEQ ID NO:681) t5-14.nt (SEQ ID NO:682) f5-14.aa (SEQ ID NO:683)t5-14.aa (SEQ ID NO:684) f5-15.nt (SEQ ID NO:685) t5-15.nt (SEQ IDNO:686) f5-15.aa (SEQ ID NO:687) t5-15.aa (SEQ ID NO:688) f51-2.nt (SEQID NO:689) t51-2.nt (SEQ ID NO:690) f51-2.aa (SEQ ID NO:691) t51-2.aa(SEQ ID NO:692) f6-21.nt (SEQ ID NO:693) t6-21.nt (SEQ ID NO:694)f6-21.aa (SEQ ID NO:695) t6-21.aa (SEQ ID NO:696) f6-27.nt (SEQ IDNO:697) t6-27.nt (SEQ ID NO:698) f6-27.aa (SEQ ID NO:699) t6-27.aa (SEQID NO:700) f6-5.nt (SEQ ID NO:701) t6-5.nt (SEQ ID NO:702) f6-5.aa (SEQID NO:703) t6-5.aa (SEQ ID NO:704) f7-30.nt (SEQ ID NO:705) t7-30.nt(SEQ ID NO:706) f7-30.aa (SEQ ID NO:707) t7-30.aa (SEQ ID NO:708)f76-1.nt (SEQ ID NO:709) t76-1.nt (SEQ ID NO:710) f76-1.aa (SEQ IDNO:711) t76-1.aa (SEQ ID NO:712) f8-10.nt (SEQ ID NO:713) t8-10.nt (SEQID NO:714) f8-10.aa (SEQ ID NO:715) t8-10.aa (SEQ ID NO:716) f8-14.nt(SEQ ID NO:717) t8-14.nt (SEQ ID NO:718) f8-14.aa (SEQ ID NO:719)t8-14.aa (SEQ ID NO:720) f01A.nt BB001 (SEQ ID NO:721) t01A.nt BB001(SEQ ID NO:722) f01A.aa BB001 (SEQ ID NO:723) t01A.aa BB001 (SEQ IDNO:724) f02A.nt BB002 (SEQ ID NO:725) t02A.nt BB002 (SEQ ID NO:726)f02A.aa BB002 (SEQ ID NO:727) t02A.aa BB002 (SEQ ID NO:728) f03A.ntBB006 (SEQ ID NO:729) t03A.nt BB006 (SEQ ID NO:730) f03A.aa BB006 (SEQID NO:731) t03A.aa BB006 (SEQ ID NO:732) f04A.nt BB011 (SEQ ID NO:733)t04A.nt BB011 (SEQ ID NO:734) f04A.aa BB011 (SEQ ID NO:735) t04A.aaBB011 (SEQ ID NO:736) f05A.nt BB009 (SEQ ID NO:737) t05A.nt BB009 (SEQID NO:738) f05A.aa BB009 (SEQ ID NO:739) t05A.aa BB009 (SEQ ID NO:740)f06A.nt BB014 (SEQ ID NO:741) t06A.nt BB014 (SEQ ID NO:742) f06A.aaBB014 (SEQ ID NO:743) t06A.aa BB014 (SEQ ID NO:744) f07A.nt BB023 (SEQID NO:745) t07A.nt BB023 (SEQ ID NO:746) f07A.aa BB023 (SEQ ID NO:747)t07A.aa BB023 (SEQ ID NO:748) f08A.nt BB024 (SEQ ID NO:749) t08A.ntBB024 (SEQ ID NO:750) f08A.aa BB024 (SEQ ID NO:751) t08A.aa BB024 (SEQID NO:752) f09A.nt BB025 (SEQ ID NO:753) t09A.nt BB025 (SEQ ID NO:754)t09A.aa BB025 (SEQ ID NO:755) t09A.aa BB025 (SEQ ID NO:756)

TABLE 2 Closest matching sequences between the polypeptides of thepresent invention and sequences in GenBank and Derwent databases. GenSeqBLAST BLAST Query Access No. GenSeq Gene Description Score P-Valuef01A.aa gi|2690256 (AE000790) antigen, P35, putative 1523 5.90E−206[Borrelia burgdorferi] f02A.aa gi|2690286 (AE000790) B. burgdorferipredicted 1320 2.10E−174 coding region BBA69 [Borrelia f02A.aagi|2690285 (AE000790) B. burgdorferi predicted 278 7.50E−71 codingregion BBA68 [Borrelia f02A.aa gi|2690105 (AE000789) B. burgdorferipredicted 151 8.40E−54 coding region BBI38 [Borrelia f02A.aa gi|2690092(AE000789) antigen, P35, putative 151 2.70E−48 [Borrelia burgdorferi]f02A.aa gi|2690183 (AE000787) antigen, P35, putative 155 4.20E−22[Borrelia burgdorferi] f02A.aa gi|2690106 (AE000789) B. burgdorferipredicted 154 1.30E−21 coding region BBI39 [Borrelia f03A.aa gi|2688051(AE001127) antigen, S2, putative 1223 7.60E−164 [Borrelia burgdorferi]f03A.aa gi|1063419 S2 gene product [Borrelia burgdorferi] 116 3.00E−22f03A.aa gi|2690227 (AE000790) antigen, S2 [Borrelia 116 9.70E−22burgdorferi] >pir|D70207|D70207 f03A.aa gi|2690128 (AE000788) proteinp23 [Borrelia 110 5.70E−19 burgdorferi] >pir|C70257|C70257 f03A.aagi|2689956 (AE000785) protein p23 [Borrelia 104 7.90E−15burgdorferi] >pir|D70225|D70225 f04A.aa gi|2690078 (AE000784) B.burgdorferi predicted 1873 5.60E−250 coding region BBH18 [Borreliaf04A.aa gi|2690192 (AE000787) B. burgdorferi predicted 167 1.40E−15coding region BBJ13 [Borrelia f05A.aa gi|2687919 (AE001117) B.burgdorferi predicted 696 4.20E−92 coding region BB0028 [Borreliaf06A.aa gi|2690129 (AE000788) outer membrane protein 884 4.80E−124[Borrelia burgdorferi] f06A.aa gi|2690089 (AE000789) conservedhypothetical 731 2.20E−118 protein [Borrelia burgdorferi] f06A.aagi|520783 unknown [Borrelia burgdorferi] 337 4.30E−58 >gi|551742 unknown[Borrelia f07A.aa gi|2688608 (AE001168) flagellar filament outer 16682.50E−224 layer protein (flaA) [Borrelia f07A.aa gi|1575447 FlaA protein[Borrelia burgdorferi] 1645 3.60E−221 >gi|1019754 orf [Borrelia f07A.aagi|152896 flagellar filament surface antigen 144 1.70E−38 [Spirochaetaaurantia] f07A.aa gi|155059 endoflagellar sheath protein 139 3.80E−28[Treponema pallidum] f07A.aa gi|433524 flagellin FlaA1 [Serpulina 1193.00E−26 hyodysenteriae] >gi|904393 endoflagellar f07A.aapir|A32814|A32814 flagellar filament surface antigen - 116 9.40E−11Spirochaeta aurantia f08A.aa gi|1209837 lipoprotein [Borreliaburgdorferi] 508 2.10E−78 f08A.aa gi|2121280 (AF000270) lipoprotein[Borrelia 547 4.00E−70 burgdorferi] >gi|3095109 f08A.aa gi|1209873lipoprotein [Borrelia burgdorferi] 303 3.70E−51 f08A.aa gi|1209843lipoprotein [Borrelia burgdorferi] 395 2.20E−49 f08A.aa gi|1209849lipoprotein [Borrelia burgdorferi] 219 2.60E−27 f08A.aa gi|3095105(AF046998) 2.9-8 lipoprotein [Borrelia 234 4.30E−27 burgdorferi] f08A.aagi|1209831 lipoprotein [Borrelia burgdorferi] 209 1.10E−22 f08A.aagi|3095107 (AF046999) 2.9-9 lipoprotein [Borrelia 200 1.80E−22burgdorferi] f08A.aa gi|1209857 lipoprotein [Borrelia burgdorferi] 2002.50E−21 f08A.aa gnl|PID|e268244 surface-exposed lipoprotein [Borrelia142 1.80E−11 afzelii] f09A.aa gi|1209843 lipoprotein [Borreliaburgdorferi] 453 8.60E−67 f09A.aa gi|2121280 (AF000270) lipoprotein[Borrelia 379 1.00E−56 burgdorferi] >gi|3095109 f09A.aa gi|1209873lipoprotein [Borrelia burgdorferi] 282 1.10E−45 f09A.aa gi|1209837lipoprotein [Borrelia burgdorferi] 357 7.10E−44 f09A.aa gi|1209849lipoprotein [Borrelia burgdorferi] 143 1.60E−13 f09A.aa gnl|PID|e268244surface-exposed lipoprotein [Borrelia 111 3.60E−13 afzelii] f09A.aagi|3095105 (AF046998) 2.9-8 lipoprotein [Borrelia 142 5.40E−13burgdorferi] f101.aa gi|2688708 (AE001176) conserved hypothetical 10994.50E−152 protein [Borrelia burgdorferi] f105.aa gi|2688693 (AE001175)B. burgdorferi predicted 1276 2.20E−177 coding region BB0758 [Borreliaf11- gi|2690139 (AE000788) B. burgdorferi predicted 1473 4.70E−193 12.aacoding region BBK01 [Borrelia f11- gi|2690030 (AE000786) B. burgdorferipredicted 1066 1.40E−138 12.aa coding region BBG01 [Borrelia f11-gi|2690074 (AE000784) B. burgdorferi predicted 173 6.20E−93 12.aa codingregion BBH37 [Borrelia f11- gi|2690188 (AE000787) B. burgdorferipredicted 192 2.70E−75 12.aa coding region BBJ08 [Borrelia f11-4.aagi|2690150 (AE000788) B. burgdorferi predicted 1144 2.70E−147 codingregion BBK12 [Borrelia f11-4.aa gi|2690145 (AE000788) B. burgdorferipredicted 852 5.70E−127 coding region BBK07 [Borrelia f11-4.aagi|2690095 (AE000789) B. burgdorferi predicted 153 1.30E−34 codingregion BBI10 [Borrelia f11-4.aa gi|2690197 (AE000787) B. burgdorferipredicted 115 1.40E−12 coding region BBJ31 [Borrelia f11-4.aa gi|2690219(AE000787) B. burgdorferi predicted 115 1.40E−12 coding region BBJ45[Borrelia f112- gi|2690054 (AE000784) B. burgdorferi predicted 5737.00E−75 1.aa coding region BBH06 [Borrelia f12.aa gi|2688785 (AE001182)B. burgdorferi predicted 6008 0 coding region BB0838 [Borrelia f129.aagi|2688685 (AE001174) B. burgdorferi predicted 987 6.20E−133 codingregion BB0739 [Borrelia f14-8.aa gi|2689955 (AE000785) antigen, P35,putative 385 2.70E−75 [Borrelia burgdorferi] f14-8.aa gi|2690120(AE000789) B. burgdorferi predicted 330 2.60E−66 coding region BBI34[Borrelia f14-8.aa gi|2690052 (AE000784) antigen, P35, putative 2874.00E−64 [Borrelia burgdorferi] f14-8.aa gi|2690100 (AE000789) B.burgdorferi predicted 172 1.10E−38 coding region BBI16 [Borreliaf14-8.aa gi|2690115 (AE000789) B. burgdorferi predicted 173 1.70E−28coding region BBI28 [Borrelia f14-8.aa gi|2690116 (AE000789) B.burgdorferi predicted 163 8.20E−24 coding region BBI29 [Borreliaf14-8.aa gi|2690207 (AE000787) B. burgdorferi predicted 220 1.90E−23coding region BBJ02 [Borrelia f14-8.aa gi|2690099 (AE000789) B.burgdorferi predicted 140 3.60E−12 coding region BBI15 [Borreliaf14-8.aa gi|2690125 (AE000788) antigen, P35, putative 111 1.00E−11[Borrelia burgdorferi] f142.aa gi|2688655 (AE001172) glutamatetransporter 2233 7.19999999999982e−311 (gltP) [Borrelia burgdorferi]f142.aa gnl|PID|e233874 hypothetical protein [Bacillus subtilis] 7272.60E−156 >gnl|PID|e1182902 f142.aa gnl|PID|d1016231Proton/sodium-glutamate symport 762 6.60E−146 protein(Glutamate-aspartate f142.aa gi|1574711 proton glutamate symport protein(gltP) 903 2.10E−131 [Haemophilus influenzae] f142.aa gi|2983758(AE000735) proton/sodium-glutamate 111 8.40E−36 symport protein [Aquifexf142.aa gi|143000 proton glutamate symport protein 125 1.20E−30[Bacillus stearothermophilus] f142.aa gi|143002 proton glutamate symportprotein 125 1.90E−28 [Bacillus caldotenax] f142.aa gnl|PID|e1183024proton/sodium-glutamate symport 122 2.20E−25 protein [Bacillus subtilis]f142.aa gnl|PID|d1022697 glutamate transporter [Caenorhabditis 1211.80E−22 elegans] f142.aa gi|1255318 coded for by C. elegans cDNAcm08h9; 121 2.10E−22 coded for by C. elegans cDNA f142.aa gi|2388712(AF017105) amino acid transporter 135 3.60E−22 [Chlamydia psittaci]f142.aa gi|2655021 (AF018259) glutamate transporter 5A 125 7.70E−22[Ambystoma tigrinum] f142.aa gnl|PID|e149542 gluT-R gene product[Clostridium 199 4.60E−21 perfringens] f142.aa gi|396412 gltP[Escherichia coli] >gi|147160 109 7.90E−21 proton-glutamate [Escherichiaf147.aa gi|2688656 (AE001172) NADH oxidase, water- 2245 7.20E−303forming (nox) [Borrelia burgdorferi] f147.aa gi|642030 NADH oxidase[Serpulina 318 9.20E−105 hyodysenteriae] f147.aa gi|2650234 (AE001077)NADH oxidase (noxA-2) 303 2.90E−93 [Archaeoglobus fulgidus] f147.aagi|2792490 (AF041467) coenzyme A disulfide 194 2.60E−90 reductase[Staphylococcus aureus] f147.aa gi|2650383 (AE001088) NADH oxidase(noxA-1) 286 3.30E−88 [Archaeoglobus fulgidus] f147.aa gnl|PID|d1009320H2O-forming NADH Oxidase 369 4.30E−85 [Streptococcus mutans] f147.aagi|49023 NADH peroxidase [Enterococcus 638 3.20E−83faecalis] >pir|S18332|S18332 NADH f147.aa gi|1591361 NADH oxidase (nox)[Methanococcus 535 4.80E−83 jannaschii] >pir|A64381|A64381 f147.aagi|2622461 (AE000898) NADH oxidase 303 8.40E−72 [Methanobacteriumthermoautotrophicum] f147.aa gi|47045 NADH oxidase [Enterococcusfaecalis] 547 8.80E−71 >pir|S26965|S26965 NADH oxidase f147.aagi|2650233 (AE001077) NADH oxidase (noxA-3) 312 2.00E−63 [Archaeoglobusfulgidus] f147.aa gi|1674132 (AE000044) Mycoplasma pneumoniae, 1757.00E−61 NADH oxidase; similar to f147.aa gi|1045969 NADH oxidase[Mycoplasma 164 4.10E−51 genitalium] >pir|D64230|D64230 NADH f147.aagi|2648692 (AE000975) NADH oxidase (noxA-5) 143 2.00E−40 [Archaeoglobusfulgidus] f147.aa gi|2983379 (AE000709) NADH oxidase [Aquifex 1625.50E−30 aeolicus] f150.aa gi|2688659 (AE001172) conserved hypothetical1319 2.70E−179 protein [Borrelia burgdorferi] f150.aa gi|2983887(AE000743) hypothetical protein 238 1.40E−25 [Aquifex aeolicus] f150.aagi|2581796 (AF001974) putative TrkA 175 5.80E−23 [Thermoanaerobacterethanolicus] f150.aa gi|1377829 unknown [Bacillus subtilis] 2121.50E−21 >gnl|PID|d1007628 orf4 [Bacillus f150.aa gnl|PID|e1185982similar to hypothetical proteins 181 6.00E−17 [Bacillus subtilis]f150.aa gnl|PID|d1011497 hypothetical protein [Synechocystis sp.] 1283.70E−11 >pir|S75999|S75999 f152.aa gi|2688660 (AE001172) K+ transportprotein (ntpJ) 2200 2.40000000001213e−313 [Borrelia burgdorferi] f152.aagi|2983882 (AE000743) K+ transport protein 239 3.60E−106 homolog[Aquifex aeolicus] f152.aa gnl|PID|e1184940 similar to Na+-transportingATP 158 6.60E−64 synthase [Bacillus subtilis] f152.aa gnl|PID|e1185983similar to Na+-transporting ATP 131 3.40E−62 synthase [Bacillussubtilis] f152.aa gnl|PID|d1018749 Na+-ATPase subunit J [Synechocystis141 1.70E−55 sp.] >pir|S75455|S75455 f152.aa gnl|PID|d1004799 Na+-ATPasesubunit J [Enterococcus 209 4.00E−45 hirae] f152.aa gi|2581795(AF001974) putative TrkG 149 2.20E−29 [Thermoanaerobacter ethanolicus]f152.aa gi|1674061 (AE000036) Mycoplasma pneumoniae, 104 4.00E−28 Na(+)translocating ATPase f152.aa gi|1046024 Na+ ATPase subunit J [Mycoplasma114 2.80E−27 genitalium] >pir|F64235|F64235 Na+ f152.aa gi|567062 HKT1[Triticum aestivum] 137 2.00E−17 >pir|S47582|S47582 high-affinitypotassium f154.aa gi|2688664 (AE001172) B. burgdorferi predicted 2456 0coding region BB0722 [Borrelia f157.aa gi|2688641 (AE001171) rodshape-determining 2300 0 protein (mreB-2) [Borrelia f157.aa gi|143657endospore forming protein [Bacillus 224 2.60E−61 subtilis] f157.aagi|580938 internal open reading frame (AA 1-290) 224 2.60E−61 [Bacillussubtilis] f157.aa gi|2982781 (AE000670) rod shape determining 3335.40E−61 protein RodA [Aquifex aeolicus] f157.aa gi|580937 spoVE geneproduct (AA 1-366) 224 7.70E−59 [Bacillus subtilis] >gnl|PID|e1185111f157.aa gi|147695 rod-shape-determining protein 340 6.10E−58[Escherichia coli] >gi|1778551 f157.aa gnl|PID|e328589 sfr [Streptomycescoelicolor] 362 6.40E−58 f157.aa gi|1572976 rod shape-determiningprotein (mreB) 307 4.00E−56 [Haemophilus influenzae] f157.aagnl|PID|e1185075 similar to cell-division protein [Bacillus 203 2.60E−45subtilis] f157.aa gi|1469784 putative cell division protein ftsW 2316.90E−45 [Enterococcus hirae] f157.aa gi|1016213 strong sequencesimilarity to FtsW, 206 3.00E−41 RodA, and SpoV-E [Cyanophora f157.aagnl|PID|d1019002 rod-shape-determining protein 184 1.60E−38[Synechocystis sp.] f157.aa gi|146039 cell division protein [Escherichiacoli] 104 8.30E−35 >gi|40857 FtsW protein f157.aa gi|1574692 celldivision protein (ftsW) 114 3.30E−33 [Haemophilus influenzae] f157.aagi|1165286 FtsW [Borrelia burgdorferi] 170 6.20E−32 >gi|2688164(AE001137) cell division f17-6.aa gi|2690100 (AE000789) B. burgdorferipredicted 1250 1.70E−164 coding region BBI16 [Borrelia f17-6.aagi|2690120 (AE000789) B. burgdorferi predicted 142 3.40E−59 codingregion BBI34 [Borrelia f17-6.aa gi|2690115 (AE000789) B. burgdorferipredicted 447 6.70E−56 coding region BBI28 [Borrelia f17-6.aa gi|2690052(AE000784) antigen, P35, putative 182 1.10E−34 [Borrelia burgdorferi]f17-6.aa gi|2689955 (AE000785) antigen, P35, putative 196 6.60E−34[Borrelia burgdorferi] f17-6.aa gi|2690114 (AE000789) B. burgdorferipredicted 176 1.00E−16 coding region BBI27 [Borrelia f17-6.aagnl|PID|d1012343 gene required for phosphoylation of 178 2.80E−15oligosaccharides/has f17-6.aa gi|2690207 (AE000787) B. burgdorferipredicted 114 3.50E−13 coding region BBJ02 [Borrelia f17-6.aagnl|PID|e329895 (AJ000496) cyclic nucleotide-gated 152 1.10E−11 channelbeta subunit f170.aa gi|2688652 (AE001171) B. burgdorferi predicted 5242.60E−73 coding region BB0708 [Borrelia f186.aa gi|2688622 (AE001169) B.burgdorferi predicted 792 1.80E−105 coding region BB0689 [Borreliaf186.aa gi|2688622 (AE001169) B. burgdorferi predicted 792 1.80E−105coding region BB0689 [Borrelia f19-2.aa gi|2690120 (AE000789) B.burgdorferi predicted 1341 2.70E−177 coding region BBI34 [Borreliaf19-2.aa gi|2689955 (AE000785) antigen, P35, putative 347 7.00E−53[Borrelia burgdorferi] f19-2.aa gi|2690052 (AE000784) antigen, P35,putative 254 7.70E−53 [Borrelia burgdorferi] f19-2.aa gi|2690100(AE000789) B. burgdorferi predicted 142 6.60E−50 coding region BBI16[Borrelia f19-2.aa gi|2690115 (AE000789) B. burgdorferi predicted 1447.60E−34 coding region BBI28 [Borrelia f19-2.aa gi|2690116 (AE000789) B.burgdorferi predicted 183 2.20E−21 coding region BBI29 [Borreliaf19-2.aa gi|2690207 (AE000787) B. burgdorferi predicted 171 2.00E−16coding region BBJ02 [Borrelia f19-2.aa gi|2690099 (AE000789) B.burgdorferi predicted 166 1.20E−15 coding region BBI15 [Borreliaf19-2.aa gi|2690125 (AE000788) antigen, P35, putative 122 5.70E−14[Borrelia burgdorferi] f19-4.aa gi|2690116 (AE000789) B. burgdorferipredicted 1129 1.30E−150 coding region BBI29 [Borrelia f19-4.aagi|2690099 (AE000789) B. burgdorferi predicted 260 3.00E−30 codingregion BBI15 [Borrelia f19-4.aa gi|2689955 (AE000785) antigen, P35,putative 180 1.80E−23 [Borrelia burgdorferi] f19-4.aa gi|2690120(AE000789) B. burgdorferi predicted 183 1.50E−21 coding region BBI34[Borrelia f19-4.aa gi|2690052 (AE000784) antigen, P35, putative 1921.20E−19 [Borrelia burgdorferi] f19-4.aa gi|2690207 (AE000787) B.burgdorferi predicted 149 8.90E−14 coding region BBJ02 [Borreliaf19-4.aa gi|2690098 (AE000789) B. burgdorferi predicted 138 8.00E−12coding region BBI14 [Borrelia f19-6.aa gi|2690115 (AE000789) B.burgdorferi predicted 995 1.20E−131 coding region BBI28 [Borreliaf19-6.aa gi|2690100 (AE000789) B. burgdorferi predicted 447 3.00E−55coding region BBI16 [Borrelia f19-6.aa gi|2689955 (AE000785) antigen,P35, putative 219 2.00E−36 [Borrelia burgdorferi] f19-6.aa gi|2690120(AE000789) B. burgdorferi predicted 144 3.50E−34 coding region BBI34[Borrelia f19-6.aa gi|2690052 (AE000784) antigen, P35, putative 1306.30E−12 [Borrelia burgdorferi] f196.aa gi|2688620 (AE001169)methyl-accepting 3093 0 chemotaxis protein (mcp-5) [Borrelia f196.aagi|2688621 (AE001169) methyl-accepting 615 1.90E−83 chemotaxis protein(mcp-4) [Borrelia f196.aa gi|496484 tlpC gene product [Bacillussubtilis] 180 6.90E−28 >pir|I40496|I40496 methylation f196.aagnl|PID|d1007002 methyl-accepting chemotaxis protein 180 4.90E−27 TlpC[Bacillus subtilis] f196.aa gnl|PID|e1173493 methyl-accepting chemotaxisprotein 162 5.10E−25 [Bacillus subtilis] f196.aa gi|882594 ORF_f506[Escherichia coli] 204 1.70E−24 >gi|1789453 (AE000389) aerotaxis f196.aagi|148350 tas [Enterobacter aerogenes] 179 1.80E−24 >pir|D32302|D32302probable aspartate f196.aa gi|1066850 putative [Rhodobacter capsulatus]207 1.80E−24 >pir|JC4735|JC4735 f196.aa gi|154381 chemoreceptor[Salmonella 230 2.00E−24 typhimurium] >pir|A47178|A47178 f196.aagi|459690 transmembrane receptor [Bacillus 212 1.40E−23subtilis] >gnl|PID|e1185997 f196.aa gi|805015 MCPA protein [Rhodobacter237 2.10E−23 sphaeroides] >pir|S70094|S54262 f196.aa gi|40424 mcpA geneproduct [Caulobacter 238 7.30E−23 crescentus] >pir|S23064|S23064 mcpAf196.aa gi|144913 sensory transducer protein [Clostridium 227 8.90E−23thermocellum] f196.aa gi|1061063 Trg sensory transducer protein 2112.40E−20 [Escherichia coli] f196.aa gnl|PID|d1015762 Methyl-acceptingchemotaxis protein III 211 2.50E−20 (MCP-III) (Ribose and f197.aagi|2688621 (AE001169) methyl-accepting 3724 0 chemotaxis protein (mcp-4)[Borrelia f197.aa gi|2688620 (AE001169) methyl-accepting 615 8.40E−83chemotaxis protein (mcp-5) [Borrelia f197.aa gi|1066850 putative[Rhodobacter capsulatus] 227 9.80E−27 >pir|JC4735|JC4735 f197.aagi|882594 ORF_f506 [Escherichia coli] 217 1.00E−26 >gi|1789453(AE000389) aerotaxis f197.aa gi|154381 chemoreceptor [Salmonella 2392.80E−25 typhimurium] >pir|A47178|A47178 f197.aa gi|496484 tlpC geneproduct [Bacillus subtilis] 202 5.10E−25 >pir|I40496|I40496 methylationf197.aa gnl|PID|d1007002 methyl-accepting chemotaxis protein 2025.10E−25 TlpC [Bacillus subtilis] f197.aa gi|2564665 (AF022807) putativemethyl accepting 212 7.20E−24 chemotaxis protein [Rhizobium f197.aagi|459691 transmembrane receptor [Bacillus 215 1.10E−23subtilis] >gnl|PID|e1185996 f197.aa gi|43218 serine chemoreceptor[Escherichia coli] 236 2.80E−23 >bbs|127562 serine f197.aa gi|537197 CGSite No. 63; alternate gene name 236 2.90E−23 cheD [Escherichia coli]f197.aa gi|148077 methyl-accepting chemotaxis protein I 236 2.90E−23[Escherichia coli] >gi|2367378 f197.aa gnl|PID|d1009948 transducer[Pseudomonas aeruginosa] 178 4.20E−23 f197.aa gi|148349 tse[Enterobacter aerogenes] 234 5.50E−23 >pir|C32302|C32302 serinetransducer f197.aa gi|2626835 chemotactic transducer [Pseudomonas 1775.70E−23 aeruginosa] f200.aa gi|2688600 (AE001168) ribose/galactose ABC1887 5.10E−266 transporter, permease protein f200.aa gnl|PID|e311453unknown [Bacillus subtilis] 283 1.50E−63 >gnl|PID|e1184234 similar tof200.aa gi|2649711 (AE001042) ribose ABC transporter, 202 1.10E−47permease protein (rbsC-1) f200.aa gi|2130609 (AF000308) putativepolytopic protein 119 2.10E−27 [Mycoplasma fermentans] f200.aagnl|PID|e311493 unknown [Bacillus subtilis] 1121.10E−18 >gnl|PID|e1184235 similar to f200.aa gi|950073 membrane formingprotein 161 5.60E−16 [Mycoplasma capricolum] >pir|S77790|S77790 f200.aagi|2688599 (AE001168) ribose/galactose ABC 108 2.00E−14 transporter,permease protein f208.aa gi|2688610 (AE001168) B. burgdorferi predicted1726 6.70E−244 coding region BB0674 [Borrelia f21-4.aa gi|1197833Bbk2.11 [Borrelia burgdorferi] 474 3.00E−70 >pir|S70531|S70531 bbk2.11protein f21-4.aa gi|2627267 ErpL [Borrelia burgdorferi] 477 6.30E−69f21-4.aa gi|1707281 putative outer membrane protein 503 6.60E−66[Borrelia burgdorferi] f21-4.aa gi|896042 OspF [Borrelia burgdorferi]503 6.60E−66 >pir|S70532|S70532 outer surface protein f21-4.aagi|1707287 putative outer membrane protein 489 3.00E−60 [Borreliaburgdorferi] f21-4.aa gi|1707290 putative outer surface protein[Borrelia 342 3.20E−49 burgdorferi] f21-4.aa gi|1663633 ErpK [Borreliaburgdorferi] 268 1.70E−48 f21-4.aa gi|466482 outer surface protein F[Borrelia 321 3.80E−38 burgdorferi] >pir|I40287|I40287 f21-4.aagi|896038 BbK2.10 precursor [Borrelia 121 3.90E−34burgdorferi] >pir|S70534|S70534 bbK2.10 f21-4.aa gi|896040 BbK2.10precursor [Borrelia 118 2.30E−33 burgdorferi] >pir|S70533|S70533 bbK2.10f21-4.aa gi|1051120 outer surface protein G [Borrelia 107 3.30E−33burgdorferi] >gi|1373118 ErpG f21-4.aa gi|2444428 (AF020657) ErpXprotein [Borrelia 118 6.00E−14 burgdorferi] f210.aa gi|2688603(AE001168) conserved hypothetical 867 2.60E−116 protein [Borreliaburgdorferi] f210.aa gi|2688604 (AE001168) chemotaxis response 7331.40E−97 regulator (cheY-3) [Borrelia f210.aa gi|1408274 CheY [Borreliaburgdorferi] 720 9.00E−96 f210.aa gi|1765976 chemotaxis protein CheY[Treponema 405 6.60E−52 pallidum] f210.aa gi|142682 chemotactic responseprotein [Bacillus 184 8.00E−30 subtilis] >gnl|PID|e1185224 f210.aagi|940149 CheY [Thermotoga maritima] 171 1.50E−27 f210.aa gi|2649557(AE001031) chemotaxis response 168 1.50E−26 regulator (cheY)[Archaeoglobus f210.aa gi|620085 cheY gene product [Listeria 1833.00E−26 monocytogenes] f210.aa gnl|PID|e249646 YneI [Bacillussubtilis] >gi|870926 166 4.00E−24 response regulator f210.aa gi|149620ORF2 [Leptospira borgpetersenii] 121 4.70E−22 >sp|P24086|YLB3_LEPINHYPOTHETICAL f210.aa gi|1408275 orfX; putative OrfX protein [Borrelia208 9.20E−22 burgdorferi] f210.aa gi|994802 cheY gene product[Halobacterium 139 8.90E−18 salinarium] >pir|S58645|S58645 CheY f210.aagi|143598 spo0F [Bacillus subtilis] >gi|143601 113 4.70E−11 Spo0Fprotein [Bacillus f216.aa gi|2688586 (AE001167) conserved hypothetical804 1.20E−109 protein [Borrelia burgdorferi] f216.aa gi|1575446 orfA[Borrelia burgdorferi] 472 1.10E−91 f219.aa gi|2688594 (AE001167) B.burgdorferi predicted 1122 3.10E−148 coding region BB0664 [Borreliaf22.aa gi|2688779 (AE001181) B. burgdorferi predicted 1400 4.90E−188coding region BB0832 [Borrelia f22.aa gi|2688779 (AE001181) B.burgdorferi predicted 1400 4.90E−188 coding region BB0832 [Borreliaf221.aa gi|2688596 (AE001167) B. burgdorferi predicted 692 2.60E−93coding region BB0662 [Borrelia f229.aa gi|2688591 (AE001167)oxygen-independent 863 7.80E−120 coproporphyrinogen III oxidase,f24-1.aa gi|2039285 putative vls recombination cassette Vls6 9241.80E−114 [Borrelia burgdorferi] f24-1.aa gi|2039284 putative vlsrecombination cassette Vls5 867 6.30E−107 [Borrelia burgdorferi]f24-1.aa gi|2039287 putative vls recombination cassette Vls8 8241.50E−104 [Borrelia burgdorferi] f24-1.aa gi|2039289 putative vlsrecombination cassette 829 7.50E−102 Vls10 [Borrelia burgdorferi]f24-1.aa gi|2039320 vmp-like sequence protein VlsE 644 1.10E−98[Borrelia burgdorferi] f24-1.aa gi|2039288 putative vls recombinationcassette Vls9 783 8.20E−96 [Borrelia burgdorferi] f24-1.aa gi|2039330vmp-like sequence protein VlsE 742 6.30E−95 [Borrelia burgdorferi]f24-1.aa gi|2039336 vmp-like sequence protein VlsE 509 1.50E−92[Borrelia burgdorferi] f24-1.aa gi|2039286 putative vls recombinationcassette Vls7 754 6.60E−92 [Borrelia burgdorferi] f24-1.aa gi|2039324vmp-like sequence protein VlsE 488 8.10E−86 [Borrelia burgdorferi]f24-1.aa gi|2039316 vmp-like sequence protein VlsE 531 1.70E−85[Borrelia burgdorferi] f24-1.aa gi|2039312 vmp-like sequence proteinVlsE 531 1.20E−83 [Borrelia burgdorferi] f24-1.aa gi|2039326 vmp-likesequence protein VlsE 476 2.00E−82 [Borrelia burgdorferi] f24-1.aagi|2039332 vmp-like sequence protein VlsE 474 5.10E−82 [Borreliaburgdorferi] f24-1.aa gi|2039328 vmp-like sequence protein VlsE 4203.50E−59 [Borrelia burgdorferi] f253.aa gi|2688567 (AE001165) Na+/H+antiporter (nhaC- 2247 0 1) [Borrelia burgdorferi] f253.aa gi|2688566(AE001165) Na+/H+ antiporter (nhaC- 609 6.40E−155 2) [Borreliaburgdorferi] f253.aa gi|2209268 Na+/H+ antiporter [Bacillus firmus] 1589.40E−15 >pir|A41594|A41594 f253.aa gi|1574661 Na+/H+ antiporter (nhaC)143 4.20E−14 [Haemophilus influenzae] f253.aa gnl|PID|e1185625 similarto Na+/H+ antiporter [Bacillus 137 1.20E−11 subtilis] f253.aagnl|PID|e324972 hypothetical protein [Bacillus subtilis] 1332.00E−11 >gnl|PID|e1182969 f265.aa gi|2688555 (AE001164) conservedhypothetical 1196 9.90E−161 protein [Borrelia burgdorferi] f269.aagi|2688560 (AE001164) B. burgdorferi predicted 1654 5.50E−226 codingregion BB0624 [Borrelia f28-2.aa gi|2690174 (AE000788) B. burgdorferipredicted 1683 2.80E−222 coding region BBK47 [Borrelia f28-2.aagi|2690161 (AE000788) B. burgdorferi predicted 1068 2.20E−163 codingregion BBK49 [Borrelia f28-3.aa gi|2690138 (AE000788) immunogenicprotein P37, 281 6.00E−48 putative [Borrelia burgdorferi] f28-3.aagi|2690127 (AE000788) immunogenic protein P37 209 3.20E−28 [Borreliaburgdorferi] f28-3.aa gi|2459605 immunogenic protein P37 [Borrelia 2084.50E−28 burgdorferi] f28-3.aa gi|2690137 (AE000788) immunogenic proteinP37, 172 5.50E−17 putative [Borrelia burgdorferi] f29.aa gi|2688764(AE001180) B. burgdorferi predicted 869 8.20E−116 coding region BB0826[Borrelia f290.aa gi|2688537 (AE001162) serine-type D-Ala-D-Ala 20461.50E−281 carboxypeptidase (dacA) f290.aa gi|143439 DD-carboxypeptidase[Bacillus subtilis] 161 6.60E−36 >pir|B42708|B42708 f290.aagnl|PID|e1185617 D-alanyl-D-alanine carboxypeptidase 161 6.60E−36(penicilin binding f290.aa gnl|PID|d1016562 Probable penicillin-bindingprotein. 131 3.30E−28 [Escherichia coli] f290.aa sp|P37604|DACD_SALTYPENICILLIN-BINDING PROTEIN 6B 135 9.10E−28 PRECURSOR f290.aa gi|1572974penicillin-binding protein 5 (dacA) 145 3.00E−27 [Haemophilusinfluenzae] f290.aa gi|580849 D-alanine carboxypeptidase [Bacillus 1704.10E−27 stearothermophilus] f290.aa gi|1778549 penicillin-bindingprotein 5 152 3.20E−26 [Escherichia coli] >gi|41212 precursor f290.aagi|142820 penicillin-binding protein 5 [Bacillus 137 4.60E−26 subtilis]f290.aa gi|410134 penicillin-binding protein [Bacillus 137 4.60E−26subtilis] >gnl|PID|e1185588 f290.aa gi|41218 precursor [Escherichiacoli] 136 1.30E−25 f290.aa gnl|PID|d1015262 Penicillin-binding protein 6precursor 136 1.30E−25 (D-alanyl-D-alanine f290.aa gi|1864022 pencillinbinding protein 4 155 5.10E−22 [Staphylococcus aureus] f290.aagnl|PID|e154145 penicillin binding protein 4 155 5.10E−22[Staphylococcus aureus] f290.aa gnl|PID|e264682 penicillin-bindingprotein 4 155 5.10E−22 [Staphylococcus aureus] f291.aa gi|2688538(AE001162) L-lactate permease (lctP) 2473 0 [Borrelia burgdorferi]f291.aa gnl|PID|e274704 lactate premease [Streptococcus iniae] 5861.20E−132 f291.aa gi|882504 ORF_f560 [Escherichia coli] 3453.60E−95 >gi|1789347 (AE000380) f560; This 560 aa f291.aa gi|2313225(AE000535) L-lactate permease (lctP) 359 1.10E−94 [Helicobacter pylori]f291.aa gi|2313224 (AE000535) L-lactate permease (lctP) 348 2.90E−93[Helicobacter pylori] f291.aa gi|404693 L-lactate permease [Escherichiacoli] 331 7.20E−82 >gi|466741 aug is 3rd start f291.aa gnl|PID|e313006hypothetical protein [Bacillus subtilis] 330 9.00E−80 >gnl|PID|e1186107f291.aa gnl|PID|d1022632 lactate permease [Bacillus subtilis] 3001.70E−61 f291.aa gnl|PID|e1182258 L-lactate permease [Bacillus subtilis]300 1.10E−60 >pir|F69649|F69649 f291.aa gnl|PID|d1009575 homologue ofL-lactate permease of E. coli 265 6.40E−56 [Bacillus f291.aa gi|2649804(AE001049) L-lactate permease (lctP) 170 1.50E−47 [Archaeoglobusfulgidus] f291.aa gnl|PID|e283914 L-lactate permease [Sulfolobus 1632.60E−44 solfataricus] f291.aa gi|1574148 L-lactate permease (lctP)[Haemophilus 173 6.00E−35 influenzae] f296.aa gi|2688517 (AE001161)chaperonin, putative 1276 4.40E−177 [Borrelia burgdorferi] f296.aagi|840643 mucZ gene product [Coxiella burnetii] 1017.90E−12 >pir|I40852|I40852 mucZ f3.aa gi|2688797 (AE001183) B.burgdorferi predicted 1604 1.40E−211 coding region BB0844 [Borreliaf30.aa gi|2688765 (AE001180) B. burgdorferi predicted 1343 2.00E−181coding region BB0825 [Borrelia f301.aa gi|2688521 (AE001161)methyl-accepting 2756 0 chemotaxis protein (mcp-3) [Borrelia f301.aagi|1805311 methyl-accepting chemotaxis protein B 211 7.00E−20 [Treponemadenticola] f301.aa gi|2688522 (AE001161) methyl-accepting 189 2.80E−18chemotaxis protein (mcp-2) [Borrelia f301.aa gi|2367665 (AF016689) Mcp-2[Treponema 189 3.50E−17 pallidum] f301.aa gi|2352917 (AF012922)methyl-accepting 187 5.70E−17 chemotaxis protein [Treponema f301.aagi|1354776 MCP-1 [Treponema pallidum] 189 5.90E−17 f301.aa gi|2619023(AF027868) YoaH [Bacillus subtilis] 184 2.80E−16 >gnl|PID|e1185333similar to f301.aa gi|1654421 transducer HtB protein [Halobacterium 1772.20E−15 salinarum] f301.aa gi|415694 chemoreceptor [Desulfovibriovulgaris] 163 3.50E−15 >pir|G36943|G36943 f301.aa gi|459691transmembrane receptor [Bacillus 163 4.90E−15subtilis] >gnl|PID|e1185996 f301.aa gi|2104730 ORF2 [Desulfurococcus sp.] 173 5.80E−15 f301.aa gi|2914132 methyl accepting chemotaxis homolog170 1.10E−14 [Treponema denticola] f301.aa gi|459689 transmembranereceptor [Bacillus 164 1.30E−14 subtilis] >gnl|PID|e1185998 f301.aagi|496484 tlpC gene product [Bacillus subtilis] 1703.80E−14 >pir|I40496|I40496 methylation f301.aa gi|2313163 (AE000530)methyl-accepting 170 6.30E−14 chemotaxis transducer (tlpC) f308.aagi|2688527 (AE001161) B. burgdorferi predicted 1227 1.70E−176 codingregion BB0592 [Borrelia f31-2.aa gi|2690202 (AE000787) B. burgdorferipredicted 1771 7.20E−235 coding region BBJ36 [Borrelia f31-2.aagi|2690200 (AE000787) B. burgdorferi predicted 423 4.60E−88 codingregion BBJ34 [Borrelia f31.aa gi|2688766 (AE001180) B. burgdorferipredicted 957 7.80E−133 coding region BB0824 [Borrelia f314.aagi|2688509 (AE001160) pfs protein (pfs-2) 1329 7.40E−180 [Borreliaburgdorferi] f314.aa gi|2690087 (AE000789) pfs protein (pfs) [Borrelia335 1.50E−77 burgdorferi] f314.aa gi|2688288 (AE001143) pfs protein(pfs-1) 266 1.00E−65 [Borrelia burgdorferi] f314.aa gi|2738591(AF012886) Pfs [Buchnera aphidicola] 115 1.70E−52 f314.aa gi|1552737similar to purine nucleoside 133 6.90E−52 phosphorylase (deoD)[Escherichia f314.aa gnl|PID|e1183957 similar to purine nucleoside 1571.20E−49 phosphorylase [Bacillus f314.aa gi|147158 pfs [Escherichiacoli] >gi|457107 ORF 133 2.50E−42 [Escherichia coli] {(SUB 9-219}f314.aa gi|1574146 pfs protein (pfs) [Haemophilus 110 2.70E−37influenzae] >pir|C64169|C64169 pfs f314.aa gi|2267164 (AF009177) pfsprotein homolog 118 3.30E−23 [Helicobacter pylori] f314.aa gi|2313168(AE000530) pfs protein (pfs) 115 1.00E−22 [Helicobacter pylori] f314.aagi|1777939 Pfs [Treponema pallidum] 102 1.90E−20 f314.aa gi|2689970(AE000785) B. burgdorferi predicted 191 1.50E−19 coding region BBE07[Borrelia f314.aa gnl|PID|e249405 unknown [Mycobacterium tuberculosis]105 7.60E−16 >sp|Q10889|Y05A_MYCTU f32-4.aa gi|2690221 (AE000787) B.burgdorferi predicted 1192 4.00E−163 coding region BBJ47 [Borreliaf32-4.aa gi|2689979 (AE000785) B. burgdorferi predicted 103 4.10E−11coding region BBE16 [Borrelia f32.aa gi|2688767 (AE001180) B.burgdorferi predicted 623 1.80E−81 coding region BB0823 [Borrelia f32.aagi|2688767 (AE001180) B. burgdorferi predicted 623 1.80E−81 codingregion BB0823 [Borrelia f320.aa gi|2688497 (AE001159) carboxypeptidase,putative 1373 6.40E−186 [Borrelia burgdorferi] f320.aa gi|2529473(AF006665) YokZ [Bacillus subtilis] 136 9.80E−28 f320.aa gi|2415396(AF015775) carboxypeptidase [Bacillus 136 1.90E−27subtilis] >gnl|PID|e1185433 f320.aa gi|1209528 D,D-carboxypeptidase[Enterococcus 148 3.30E−16 faecalis] >sp|Q47746|VANY_ENTFA f320.aagi|155044 vanY [Transposon Tn1546] >gi|149126 142 1.60E−13D,D-carboxypeptidase [Plasmid f328.aa gi|2688502 (AE001159) CTP synthase(pyrG) 869 6.10E−119 [Borrelia burgdorferi] f328.aa gi|1591801 CTPsynthase (pyrG) [Methanococcus 325 6.20E−59jannaschii] >pir|E64446|E64446 f328.aa gi|2650385 (AE001088) CTPsynthase (pyrG) 304 4.20E−54 [Archaeoglobus fulgidus] f328.aa gi|1399854CTP synthetase [Synechococcus 313 3.30E−52PCC7942] >sp|Q54775|PYRG_SYNP7 CTP f328.aa gnl|PID|d1019032 CTPsynthetase [Synechocystis sp.] 295 1.80E−50 >pir|S75840|S75840 CTPf328.aa gi|143597 CTP synthetase [Bacillus subtilis] 2741.60E−49 >gi|853762 CTP synthase [Bacillus f328.aa gi|2983754 (AE000735)CTP synthetase [Aquifex 271 1.50E−46 aeolicus] f328.aa gi|1574630 CTPsynthetase (pyrG) [Haemophilus 234 1.90E−44influenzae] >pir|F64181|F64181 f328.aa gi|413755 CTP synthetase[Spiroplasma citri] 231 3.00E−44 >sp|P52200|PYRG_SPICI CTP f328.aagi|2621483 (AE000826) CTP synthase 257 2.80E−40 [Methanobacteriumthermoautotrophicum] f328.aa gi|950067 CTP synthase [Mycoplasma 2204.10E−39 capricolum] >pir|S77767|S77767 CTP synthase f328.aa gi|904007cytidine triphosphate synthetase 219 2.00E−38 precursor [Giardiaintestinalis] f328.aa gi|147478 CTP synthetase (EC 6.3.4.2) 217 2.90E−38[Escherichia coli] f328.aa gi|882674 CTP synthetase [Escherichia coli]214 7.70E−38 >gi|1789142 (AE000361) CTP f328.aa gi|38688 CTP synthase[Azospirillum brasilense] 132 3.20E−37 >pir|I39496|S25101 CTP f342.aagi|2688495 (AE001158) B. burgdorferi predicted 944 5.30E−130 codingregion BB0563 [Borrelia f346.aa gi|1272356 phosphotransferase enzyme II[Borrelia 828 1.10E−108 burgdorferi] >gi|2688474 f346.aa gi|145603 PTSenzyme III glc [Escherichia coli] 385 8.80E−53 >gi|145605 PTS enzyme IIIglc f346.aa gi|1314675 glucose-specific component IIA of the 3859.30E−53 PTS system [Escherichia coli] f346.aa gi|47658 III(Glc) (crr)(AA 1-169) [Salmonella 382 2.30E−52 typhimurium] f346.aa gi|1574566glucose phosphotransferase enzyme III- 397 8.70E−50 glc (crr)[Haemophilus f346.aa gi|43819 nagE gene product [Klebsiella 349 2.80E−41pneumoniae] >pir|S18607|S18607 f346.aa gi|146913 N-acetylglucosaminetransport protein 334 3.20E−39 [Escherichia coli] f346.aa gi|1072418glcA [Staphylococcus carnosus] 317 7.20E−37 >pir|S46952|S46952 f346.aagi|1072419 glcB [Staphylococcus carnosus] 3151.40E−36 >pir|S63606|S46953 f346.aa gi|1146177 phosphotransferase systemglucose- 295 7.30E−36 specific enzyme II [Bacillus f346.aa gi|529001 PTSglucose-specific permease 294 8.80E−36 [Bacillus stearothermophilus]f346.aa gnl|PID|e1182187 alternate gene name: yzfA; similar to 2931.40E−33 phosphotransferase f346.aa gi|580912 enzyme III-glucose[Bacillus subtilis] 257 1.20E−30 f346.aa gi|602681 phosphocarrierprotein (enzyme IIA) 243 1.00E−28 [Mycoplasma capricolum] f346.aagi|1432153 cellobiose-specific PTS permease 257 1.20E−28 [Klebsiellaoxytoca] f352.aa gi|2688482 (AE001157) B. burgdorferi predicted 2547 0coding region BB0553 [Borrelia f352.aa gi|2688482 (AE001157) B.burgdorferi predicted 1005 1.30E−132 coding region BB0553 [Borreliaf363.aa gi|2688468 (AE001156) B. burgdorferi predicted 1109 5.40E−153coding region BB0543 [Borrelia f368.aa gi|2688450 (AE001155) conservedhypothetical 1133 4.10E−157 integral membrane protein f368.aa gi|1787004(AE000181) o234; This 234 aa ORF is 417 1.40E−67 26 pct identical (15gaps) to f368.aa gi|2314055 (AE000601) conserved hypothetical 1293.50E−16 integral membrane protein f368.aa gnl|PID|e1289272 S1R [Cowpoxvirus] 135 1.80E−14 f368.aa gnl|PID|d1003176 24 K membrane protein[Pseudomonas 108 9.00E−13 aeruginosa] f368.aa gi|41284 put. 23.5-kdprotein [Escherichia coli] 101 1.00E−11 >gi|1787205 (AE000199) f371.aagi|2688452 (AE001155) conserved hypothetical 1066 3.60E−143 protein[Borrelia burgdorferi] f371.aa gi|2196997 Orf256 [Treponema pallidum]154 1.10E−15 f373.aa gi|2688453 (AE001155) zinc protease, putative 36630 [Borrelia burgdorferi] f373.aa gi|1574200 hypothetical [Haemophilusinfluenzae] 295 2.70E−67 >pir|E64171|E64171 f373.aa gi|1787770(AE000246) f931; residues 5-650 are 289 1.10E−57 99 pct identical toYDDC_ECOLI f373.aa gi|535004 cds106 gene product [Escherichia coli] 2893.20E−57 f373.aa gi|799369 metalloendopeptidase [Pisum sativum] 1487.10E−28 f373.aa gi|2827039 (AF008444) chloroplast processing 1501.70E−26 enzyme [Arabidopsis thaliana] f373.aa gi|2983709 (AE000732)processing protease 136 4.30E−24 [Aquifex aeolicus] f373.aa gi|2314155(AE000609) protease (pqqE) 115 5.30E−23 [Helicobacterpylori] >pir|D64646|D64646 f378.aa gi|2688458 (AE001155) B. burgdorferipredicted 1030 1.30E−136 coding region BB0531 [Borrelia f384.aagi|2688435 (AE001154) inositol monophosphatase 1470 3.80E−201 [Borreliaburgdorferi] f4-15.aa gi|2690238 (AE000790) surface lipoprotein P27 14001.50E−185 [Borrelia burgdorferi] f4-15.aa gi|144008 P27 [Borreliaburgdorferi] 462 2.40E−96 >pir|S34995|S34995 surface lipoproteinf4-50.aa gi|2690243 (AE000790) decorin binding protein B 900 6.30E−117(dbpB) [Borrelia burgdorferi] f4-50.aa gi|2062381 decorin bindingprotein B [Borrelia 897 1.60E−116 burgdorferi] f4-50.aa gi|2809217(AF042796) putative decorin-binding 887 3.60E−115 protein precursor[Borrelia f4-50.aa gi|2809218 (AF042796) decorin-binding protein 1722.00E−33 precursor [Borrelia burgdorferi] f4-50.aa gi|2690249 (AE000790)decorin binding protein A 176 9.50E−33 (dbpA) [Borrelia burgdorferi]f4-50.aa gi|2062379 decorin binding protein A [Borrelia 177 6.10E−32burgdorferi] f4-66.aa gi|2690229 (AE000790) chpAI protein, putative 8071.60E−107 [Borrelia burgdorferi] f4.aa gi|2688787 (AE001183) conservedhypothetical 2408 0 integral membrane protein f4.aa gi|2697115(AF008219) unknown [Borrelia afzelii] 1138 1.90E−305 f4.aa gi|1573583 H.influenzae predicted coding region 337 2.10E−109 HI0594 [Haemophilusf4.aa gi|1788636 (AE000319) o513; This 513 aa ORF is 327 9.10E−80 31 pctidentical (30 gaps) to f4.aa gnl|PID|d1009571 homologue of hypotheticalprotein 357 5.40E−69 HI10594 of H. influenzae f42-1.aa gi|2689993(AE000794) conserved hypothetical 495 2.70E−62 protein [Borreliaburgdorferi] f42-1.aa gi|2689934 (AE000793) conserved hypothetical 3121.00E−37 protein [Borrelia burgdorferi] f43-3.aa gi|1209843 lipoprotein[Borrelia burgdorferi] 546 1.50E−69 f43-3.aa gi|2121280 (AF000270)lipoprotein [Borrelia 442 1.80E−55 burgdorferi] >gi|3095109 f43-3.aagi|1209837 lipoprotein [Borrelia burgdorferi] 365 3.10E−55 f43-3.aagi|1209873 lipoprotein [Borrelia burgdorferi] 269 5.30E−32 f43-3.aagi|1209849 lipoprotein [Borrelia burgdorferi] 141 1.70E−13 f43-3.aagi|3095105 (AF046998) 2.9-8 lipoprotein [Borrelia 140 9.60E−13burgdorferi] f43-3.aa gi|3095107 (AF046999) 2.9-9 lipoprotein [Borrelia132 1.40E−11 burgdorferi] f43.aa gi|2688752 (AE001179) B. burgdorferipredicted 2337 6.60000000084856e−315 coding region BB0811 [Borreliaf446.aa gi|2688383 (AE001151) B. burgdorferi predicted 920 7.20E−124coding region BB0464 [Borrelia f45-2.aa gi|1699017 ErpB2 [Borreliaburgdorferi] 364 7.50E−78 >gi|1373133 ErpB [Borrelia f45-2.aa gi|2627270ErpJ [Borrelia burgdorferi] 364 2.50E−77 f45-2.aa gi|2627268 ErpM[Borrelia burgdorferi] 452 9.70E−60 f45-2.aa gi|1373144 ErpD [Borreliaburgdorferi] 316 1.60E−58 f45-2.aa gi|2444428 (AF020657) ErpX protein[Borrelia 380 2.80E−55 burgdorferi] f45-2.aa gi|1051120 outer surfaceprotein G [Borrelia 213 7.10E−35 burgdorferi] >gi|1373118 ErpG f45-2.aagi|1663633 ErpK [Borrelia burgdorferi] 152 1.60E−21 f45-2.aagnl|PID|e329895 (AJ000496) cyclic nucleotide-gated 198 2.80E−16 channelbeta subunit f45-2.aa gi|466482 outer surface protein F [Borrelia 1115.70E−14 burgdorferi] >pir|I40287|I40287 f45-2.aa gi|2246532 ORF 73,contains large complex repeat 174 5.90E−14 CR 73 [Kaposi's f45-2.aagi|160299 glutamic acid-rich protein [Plasmodium 169 1.00E−13falciparum] f45-2.aa gi|1707287 putative outer membrane protein 1012.20E−13 [Borrelia burgdorferi] f45-2.aa gi|1633572 Herpesvirus saimiriORF73 homolog 175 4.10E−13 [Kaposi's sarcoma-associated f45-2.aagnl|PID|d1012343 gene required for phosphoylation of 166 5.60E−13oligosaccharides/has f45-2.aa gi|2690100 (AE000789) B. burgdorferipredicted 161 2.70E−12 coding region BBI16 [Borrelia f457.aa gi|2688369(AE001150) B. burgdorferi predicted 1021 6.20E−139 coding region BB0456[Borrelia f469.aa gi|2688368 (AE001150) Na+/H+ antiporter (napA) 15441.10E−211 [Borrelia burgdorferi] f47-2.aa gi|1209849 lipoprotein[Borrelia burgdorferi] 742 2.30E−97 f47-2.aa gi|1209857 lipoprotein[Borrelia burgdorferi] 407 7.80E−86 f47-2.aa gi|1209831 lipoprotein[Borrelia burgdorferi] 393 5.00E−82 f47-2.aa gnl|PID|e268245surface-exposed lipoprotein [Borrelia 321 2.60E−73 burgdorferi] f47-2.aagi|1209874 lipoprotein [Borrelia burgdorferi] 348 1.10E−64 f47-2.aagnl|PID|e268239 surface-exposed lipoprotein [Borrelia 333 1.40E−57garinii] f47-2.aa gnl|PID|e268244 surface-exposed lipoprotein [Borrelia292 9.60E−44 afzelii] f47-2.aa gi|3095107 (AF046999) 2.9-9 lipoprotein[Borrelia 328 3.80E−40 burgdorferi] f47-2.aa gnl|PID|e268242surface-exposed lipoprotein [Borrelia 320 1.70E−39 garinii] f47-2.aagi|1209837 lipoprotein [Borrelia burgdorferi] 210 4.80E−29 f47-2.aagi|2121280 (AF000270) lipoprotein [Borrelia 205 1.10E−27burgdorferi] >gi|3095109 f47-2.aa gi|3095105 (AF046998) 2.9-8lipoprotein [Borrelia 217 6.30E−25 burgdorferi] f47-2.aa gi|1209873lipoprotein [Borrelia burgdorferi] 113 2.40E−11 f477.aa gi|2688350(AE001149) fructose-bisphosphate 1506 3.60E−202 aldolase (fba) [Borreliaf477.aa gi|882454 fructose 1,6-bisphosphate aldolase 651 1.10E−131[Escherichia coli] >gi|41423 f477.aa gi|2708661 (AF037440) fructose1,6-bisphosphate 593 1.40E−124 aldolase [Edwardsiella f477.aa gi|1573507fructose-bisphosphate aldolase (fba) 560 8.50E−120 [Haemophilusinfluenzae] f477.aa gi|671841 fructose 1,6-bisphosphate aldolase 8563.80E−113 [Campylobacter jejuni] f477.aa gnl|PID|d1004756 fructose1,6-bisphosphate aldolase 749 1.70E−98 [Schizosaccharomyces f477.aagi|433637 yeast fructose-bisphate-aldolase 459 1.20E−92 [Saccharomycescerevisiae] >gi|3696 f477.aa gnl|PID|e190134 fructose-1,6-bisphosphatealdolase 701 6.30E−92 [Euglena gracilis] f477.aa gi|1334980 fructose 1,6bisphosphate-aldolase 647 1.50E−84 [Neurospora crassa] f477.aa gi|40495fructose-bisphosphate aldolase 204 6.80E−37 [Corynebacterium glutamicum]f477.aa gnl|PID|e315480 Fba [Mycobacterium tuberculosis] 207 1.50E−35f477.aa gi|1045692 fructose-bisphosphate aldolase 108 2.10E−23[Mycoplasma genitalium] f477.aa gnl|PID|d1003809 hypothetical protein[Bacillus subtilis] 102 2.70E−15 >gnl|PID|e1184692 f488.aa gi|2688338(AE001148) DNA gyrase, subunit A 3222 0 (gyrA) [Borrelia burgdorferi]f488.aa gi|1790876 DNA gyrase subunit A [Clostridium 822 1.80E−171acetobutylicum] f488.aa gi|2650163 (AE001072) DNA gyrase, subunit A 4831.10E−162 (gyrA) [Archaeoglobus fulgidus] f488.aa gi|40019 ORF 821 (aa1-821) [Bacillus subtilis] 836 6.10E−159 >gnl|PID|d1005785 A subunit off488.aa gi|459929 gyrase A subunit [Pseudomonas 418 7.00E−155aeruginosa] >sp|P48372|GYRA_PSEAE DNA f488.aa gi|144206 DNA gyrase A[Campylobacter jejuni] 508 7.50E−154 >pir|A48902|A48902 DNA gyrasef488.aa gi|466275 gyrase A [Mycobacterium tuberculosis] 3953.50E−152 >sp|Q07702|GYRA_MYCTU DNA f488.aa gnl|PID|e266924 GyrA[Mycobacterium tuberculosis] 395 2.00E−151 f488.aa gi|43485 DNA gyrase Asubunit [Haloferax] 275 6.10E−151 >pir|S30571|S30571 DNA topoisomerasef488.aa gnl|PID|d1025098 (AB010081) A subunit of DNA gyrase 5491.20E−150 [Bacillus sp.] f488.aa gnl|PID|e214031 DNA gyrase subunit A[Mycobacterium 388 5.90E−150 smegmatis] f488.aa gi|2731385 DNA gyrase[Serratia marcescens] 378 6.00E−148 f488.aa gnl|PID|e137038 DNAtopoisomerase (ATP-hydrolysing) 388 7.30E−147 [Mycobacterium smegmatis]f488.aa gi|41634 gyrA gene product (AA 1-875) 383 2.40E−146 [Escherichiacoli] >gi|41636 DNA gyrase f488.aa gi|497648 DNA gyrase subunit A[Mycoplasma 514 5.20E−146 genitalium] f49-2.aa gi|2039282 putative vlsrecombination cassette Vls3 943 2.30E−120 [Borrelia burgdorferi]f49-2.aa gi|2547241 vmp-like sequence protein VlsE 434 4.10E−106[Borrelia burgdorferi] f49-2.aa gi|2039324 vmp-like sequence proteinVlsE 458 3.00E−104 [Borrelia burgdorferi] f49-2.aa gi|2039281 putativevls recombination cassette Vls2 793 1.80E−100 [Borrelia burgdorferi]f49-2.aa gi|2039283 putative vls recombination cassette Vls4 7294.60E−92 [Borrelia burgdorferi] f49-2.aa gi|2039308 vmp-like sequenceprotein VlsE 652 1.40E−88 [Borrelia burgdorferi] f49-2.aa gi|2039288putative vls recombination cassette Vls9 352 1.80E−88 [Borreliaburgdorferi] f49-2.aa gi|2039332 vmp-like sequence protein VlsE 5504.40E−88 [Borrelia burgdorferi] f49-2.aa gi|2039328 vmp-like sequenceprotein VlsE 629 1.50E−85 [Borrelia burgdorferi] f49-2.aa gi|2039336vmp-like sequence protein VlsE 460 1.40E−82 [Borrelia burgdorferi]f49-2.aa gi|2039318 vmp-like sequence protein VlsE 367 6.20E−82[Borrelia burgdorferi] f49-2.aa gi|2039320 vmp-like sequence proteinVlsE 449 1.80E−77 [Borrelia burgdorferi] f49-2.aa gi|2483796 VlsE1[Borrelia burgdorferi] 497 8.20E−76 f49-2.aa gi|2039326 vmp-likesequence protein VlsE 427 2.50E−64 [Borrelia burgdorferi] f49-2.aagi|2039291 putative vls recombination cassette 409 1.30E−47 Vls13[Borrelia burgdorferi] f494.aa gi|2688346 (AE001148) B. burgdorferipredicted 547 8.20E−74 coding region BB0428 [Borrelia f5-14.aagi|2627268 ErpM [Borrelia burgdorferi] 1836 2.60E−236 f5-14.aagi|1373144 ErpD [Borrelia burgdorferi] 543 4.40E−87 f5-14.aa gi|2627270ErpJ [Borrelia burgdorferi] 503 4.30E−83 f5-14.aa gi|1699017 ErpB2[Borrelia burgdorferi] 503 2.60E−82 >gi|1373133 ErpB [Borrelia f5-14.aagi|2444428 (AF020657) ErpX protein [Borrelia 399 9.30E−57 burgdorferi]f5-14.aa gnl|PID|e329895 (AJ000496) cyclic nucleotide-gated 228 1.50E−20channel beta subunit f5-14.aa gnl|PID|d1012343 gene required forphosphoylation of 203 8.70E−18 oligosaccharides/has f5-14.aa gi|2246532ORF 73, contains large complex repeat 197 3.30E−17 CR 73 [Kaposi'sf5-14.aa gi|1633572 Herpesvirus saimiri ORF73 homolog 192 1.20E−16[Kaposi's sarcoma-associated f5-14.aa gi|3068583 (AF000580) Rep-like[Dictyostelium 197 3.60E−16 discoideum] f5-14.aa gi|2690100 (AE000789)B. burgdorferi predicted 183 2.90E−15 coding region BBI16 [Borreliaf5-14.aa gi|1825739 No definition line found 168 1.60E−14[Caenorhabditis elegans] f5-14.aa gi|3044185 (AF056936) matureparasite-infected 166 2.00E−14 erythrocyte surface antigen f5-14.aagnl|PID|e349084 E02A10.2 [Caenorhabditis elegans] 176 2.30E−14 f5-14.aagi|1051120 outer surface protein G [Borrelia 157 3.30E−12burgdorferi] >gi|1373118 ErpG f5-15.aa gi|2627267 ErpL [Borreliaburgdorferi] 1152 4.40E−147 f5-15.aa gi|1197833 Bbk2.11 [Borreliaburgdorferi] 856 3.30E−108 >pir|S70531|S70531 bbk2.11 protein f5-15.aagi|896042 OspF [Borrelia burgdorferi] 325 1.00E−72 >pir|S70532|S70532outer surface protein f5-15.aa gi|1707281 putative Outer membraneprotein 323 1.80E−72 [Borrelia burgdorferi] f5-15.aa gi|1707287 putativeouter membrane protein 322 6.60E−70 [Borrelia burgdorferi] f5-15.aagi|466482 outer surface protein F [Borrelia 448 6.80E−68burgdorferi] >pir|I40287|I40287 f5-15.aa gi|1707290 putative outersurface protein [Borrelia 290 1.90E−52 burgdorferi] f5-15.aa gi|1663633ErpK [Borrelia burgdorferi] 172 8.70E−43 f5-15.aa gi|896038 BbK2.10precursor [Borrelia 153 1.10E−42 burgdorferi] >pir|S70534|S70534 bbK2.10f5-15.aa gi|896040 BbK2.10 precursor [Borrelia 124 4.30E−39burgdorferi] >pir|S70533|S70533 bbK2.10 f5-15.aa gi|1051120 outersurface protein G [Borrelia 105 3.10E−23 burgdorferi] >gi|1373118 ErpGf5-15.aa gi|1373144 ErpD [Borrelia burgdorferi] 103 1.10E−14 f50.aagi|2688754 (AE001179) B. burgdorferi predicted 2651 0 coding regionBB0806 [Borrelia f502.aa gi|2688313 (AE001146) sensory transduction 75700 histidine kinase, putative f502.aa gnl|PID|d1025877 (AB006363)homologue of histidine 296 3.80E−58 kinase [Candida albicans] f502.aagi|1354473 Os-1p [Neurospora crassa] 275 3.30E−57 f502.aa gi|1679757two-component histidine kinase CHK-1 382 4.20E−57 [Glomerella cingulata]f502.aa gi|1262208 Nik-1 [Neurospora crassa] >gi|1262210 273 6.30E−57Nik-1 [Neurospora crassa] f502.aa gi|2460283 (AF024654) hybrid histidinekinase 273 3.90E−55 DHKB [Dictyostelium discoideum] f502.aagnl|PID|d1017789 sensory transduction histidine kinase 288 8.50E−54[Synechocystis sp. ] f502.aa gi|2623815 (AF030352) two component sensor252 4.00E−52 [Pseudomonas aeruginosa] f502.aa gi|939724 putative sensorkinase; regulatory 252 1.80E−50 protein for production of f502.aagi|151329 regulatory protein [Pseudomonas 248 1.20E−49syringae] >sp|P48027|LEMA_PSESY f502.aa pir|B41863|B41863 two-componentregulatory protein lemA - 248 1.30E−49 Pseudomonas syringae f502.aagnl|PID|d1018725 sensory transduction histidine kinase 252 2.10E−49[Synechocytis sp. ] f502.aa gnl|PID|d1002185 sensor-regulator protein[Escherichia 262 6.20E−49 coli] >gi|1789149 f502.aa gi|463195 pectatelyase [Pseudomonas viridiflava] 247 7.50E−49 f502.aa gnl|PID|d1018731sensory transduction histidine kinase 244 1.00E−48 [Synechocystis sp. ]f51-2.aa gi|2444428 (AF020657) ErpX protein [Borrelia 1755 2.20E−227burgdorferi] f51-2.aa gi|2627268 ErpM [Borrelia burgdorferi] 3993.20E−57 f51-2.aa gi|1373144 ErpD [Borrelia burgdorferi] 282 2.20E−50f51-2.aa gi|2627270 ErpJ [Borrelia burgdorferi] 271 6.00E−34 f51-2.aagi|1699017 ErpB2 [Borrelia burgdorferi] 271 2.50E−33 >gi|1373133 ErpB[Borrelia f51-2.aa gi|1051120 outer surface protein G [Borrelia 1093.70E−22 burgdoferi] >gi|1373118 ErpG f51-2.aa gnl|PID|d1012343 generequired for phosphoylation of 203 5.40E−18 oligosaccharides/hasf51-2.aa gi|1707287 putative outer membrane protein 111 7.50E−18[Borrelia burgdoferi] f51-2.aa gi|896042 OspF [Borrelia burgdorferi] 1112.10E−17 >pir|S70532|S70532 outer surface protein f51-2.aa gi|1707281putative outer membrane protein 111 7.50E−17 [Borrelia burgdorferi]f51-2.aa gnl|PID|e329895 (AJ000496) cyclic nucleotide-gated 198 1.60E−16channel beta subunit f51-2.aa gi|2246532 ORF 73, contains large complexrepeat 176 2.30E−14 CR 73 [Kaposi's f51-2.aa gnl|PID|e349084 E02A10.2[Caenorhabditis elegans] 170 2.10E−13 f51-2.aa gi|160299 glutamicacid-rich protein [Plasmodium 157 7.30E−12 falciparum] f516.aagi|2688326 (AE001146) B. burgdorferi predicted 1096 2.00E−150 codingregion BB0409 [Borrelia f517.aa gi|2688320 (AE001146) PTS system,fructose- 1637 2.30E−228 specific IIABC component (fruA-1) f517.aagnl|PID|e1183221 similar to fructose phosphotransferase 256 4.00E−88system enzyme II f517.aa gi|396296 similar to phosphotransferase system305 9.10E−86 enzyme II [Escherichia coli] f517.aa gi|405893fructose-specific IIBC component 224 4.30E−84 [Escherichiacoli] >gi|450372 f517.aa gi|151932 fructose enzyme II [Rhodobacter 2224.70E−79 capsulatus] >gi|46021 fructose f517.aa gi|1573422fructose-permease IIBC component 225 6.90E−69 (fruA) [Haemophilusinfluenzae] f517.aa gi|2688554 (AE001164) PTS system, fructose- 2368.20E−66 specific IIABC component (fruA-2) f517.aa gnl|PID|e1185030phosphotransferase system (PTS) 195 2.80E−65 fructose-specific enzymeIIBC f517.aa gi|155369 PTS enzyme-II fructose [Xanthomonas 187 8.10E−62campestris] >pir|B40944|B40944 f517.aa gi|305003 similar tofructose-specific 145 1.90E−39 phosphotransferase enzyme II f517.aagnl|PID|d1011544 HrsA [Escherichia coli] >gi|1786951 148 2.80E−39(AE000176) f517.aa gi|1813488 phosphotransferase enzyme II [Bacillus 2263.90E−39 firmus] f517.aa gi|757734 fruA gene product [Bacillus 1772.50E−36 amyloliquefaciens] >pir|S59965|S59965 f517.aa gnl|PID|d1016984PTS SYSTEM, FRUCTOSE-SPECIFIC 173 1.10E−34 IIBC COMPONENT (EIIBC-FRU)f517.aa gi|1673731 (AE000010) Mycoplasma pneumoniae, 143 9.00E−33fructose-permease IIBC component; f519.aa gi|2688327 (AE001146) B.burgdorferi predicted 1060 5.70E−145 coding region BB0406 [Borreliaf519.aa gi|2688328 (AE001146) B. burgdorferi predicted 261 1.20E−47coding region BB0405 [Borrelia f520.aa gi|2688328 (AE001146) B.burgdorferi predicted 1022 3.90E−138 coding region BB0405 [Borreliaf520.aa gi|2688327 (AE001146) B. burgdorferi predicted 261 4.00E−47coding region BB0406 [Borrelia f523.aa gi|2688300 (AE001145) glutamatetransporter, 2007 9.90E−284 putative [Borrelia burgdorferi] f526.aagi|2688309 (AE001145) B. burgdorferi predicted 1087 1.60E−145 codingregion BB0399 [Borrelia f527.aa gi|2688310 (AE001145) B. burgdorferipredicted 1814 7.60E−249 coding region BB0398 [Borrelia f541.aagi|508421 antigen P39 [Borrelia burgdorferi] 1706 5.40E−230 >gi|2688281(AE001143) basic f541.aa gi|1753225 BmpA protein [Borrelia burgdorferi]1698 6.80E−229 f541.aa gnl|PID|e1172833 bmpA(p39, ORF1) [Borrelia 16951.70E−228 burgdorferi] f541.aa gnl|PID|e1172835 membrane protein A[Borrelia 1642 3.40E−221 burgdorferi] >gi|516592 membrane f541.aagnl|PID|e1172834 membrane protein A [Borrelia 1638 1.20E−220burgdorferi] f541.aa gnl|PID|e1172828 bmpA(p39, ORF1) [Borrelia 15511.00E−208 burgdorferi] f541.aa gnl|PID|e1172829 membrane protein A[Borrelia afzelii] 1502 5.60E−202 f541.aa gnl|PID|e1172831 membraneprotein A [Borrelia afzelii] 1499 1.40E−201 f541.aa gnl|PID|e1172837membrane protein A [Borrelia garinii] 1496 3.70E−201 f541.aagnl|PID|e1172830 membrane protein A [Borrelia afzelii] 1493 9.60E−201f541.aa gnl|PID|e1172838 membrane protein A [Borrelia garinii] 14884.60E−200 f541.aa gnl|PID|e237214 membrane protein A [Borrelia garinii]1216 1.20E−162 f541.aa gnl|PID|e237209 membrane protein A [Borreliagarinii] 1211 5.90E−162 f541.aa gnl|PID|e237236 membrane protein A[Borrelia garinii] 1098 2.00E−146 f541.aa gi|2688282 (AE001143) basicmembrane protein B 518 1.20E−123 (bmpB) [Borrelia burgdorferi] f542.aagi|508422 [Borrelia burgdorferi immunodominant 711 1.70E−95 antigen P39gene, complete f542.aa gi|2688282 (AE001143) basic membrane protein B711 1.70E−95 (bmpB) [Borrelia burgdorferi] f542.aa gi|551744 membranelipoprotein [Borrelia 708 8.60E−95 burgdorferi] f542.aa gnl|PID|e1172836bmpB(p39, ORF2) [Borrelia 699 8.20E−94 burgdorferi] f542.aagnl|PID|e1172832 bmpB(p39, ORF2) [Borrelia afzelii] 634 1.00E−84 f542.aagnl|PID|e1172839 bmpB(p39, ORF2) [Borrelia garinii] 613 9.20E−82 f542.aagnl|PID|e237209 membrane protein A [Borrelia garinii] 153 1.70E−32f542.aa gnl|PID|e1172828 bmpA(p39, ORF1) [Borrelia 144 3.80E−32burgdorferi] f542.aa gnl|PID|e237214 membrane protein A [Borreliagarinii] 153 2.00E−31 f542.aa gi|1753225 BmpA protein [Borreliaburgdorferi] 155 2.80E−31 f542.aa gnl|PID|e1172833 bmpA(p39, ORF1)[Borrelia 155 2.80E−31 burgdorferi] f542.aa gi|508421 antigen P39[Borrelia burgdorferi] 155 2.80E−31 >gi|2688281 (AE001143) basic f542.aagnl|PID|e1172837 membrane protein A [Borrelia garinii] 156 1.00E−30f542.aa gnl|PID|e1172829 membrane protein A [Borrelia afzelii] 1441.90E−30 f542.aa gnl|PID|e1172830 membrane protein A [Borrelia afzelii]144 2.70E−30 f544.aa gi|2688284 (AE001143) Mg2+ transport protein 8604.20E−119 (mgtE) [Borrelia burgdorferi] f544.aa gi|1753228 MgtE[Borrelia burgdorferi] 855 2.20E−118 f544.aa gi|619724 MgtE [Bacillusfirmus] 176 3.70E−37 >pir|I40201|I40201 mgtE protein - Bacillus f544.aagi|780282 extended ORF of mgtE gene; 182 1.30E−34 transcription fromthis start point is f544.aa gnl|PID|e315479 unknown [Mycobacteriumtuberculosis] 183 4.50E−31 f544.aa gnl|PID|d1018132 Mg2+ transporter[Synechocystis sp. ] 165 4.60E−31 >pir|S77552|S77552 Mg2+ f544.aagnl|PID|e1181529 (AJ002571) YkoK [Bacillus subtilis] 1422.30E−30 >gnl|PID|e1183350 similar f544.aa gi|2621701 (AE000843) Mg2+transporter 142 3.20E−21 [Methanobacterium thermoautotrophicum] f545.aagi|2688284 (AE001143) Mg2+ transport protein 860 4.20E−119 (mgtE)[Borrelia burgdorferi] f545.aa gi|1753228 MgtE [Borrelia burgdorferi]855 2.20E−118 f545.aa gi|619724 MgtE [Bacillus firmus] 1763.70E−37 >pir|I40201|I40201 mgtE protein - Bacillus f545.aa gi|780282extended ORF of mgtE gene; 182 1.30E−34 transcription from this startpoint is f545.aa gnl|PID|e315479 unknown [Mycobacterium tuberculosis]183 4.50E−31 f545.aa gnl|PID|d1018132 Mg2+ transporter [Synechocystissp. ] 165 4.60E−31 >pir|S77552|S77552 Mg2+ f545.aa gnl|PID|e1181529(AJ002571) YkoK [Bacillus subtilis] 142 2.30E−30 >gnl|PID|e1183350similar f545.aa gi|2621701 (AE000843) Mg2+ transporter 142 3.20E−21[Methanobacterium thermoautotrophicum] f561.aa gi|49245 lipoprotein[Borrelia burgdorferi] 1000 1.30E−132 >gi|2688271 (AE001142) lipoproteinf561.aa gi|495738 P22 [Borrelia burgdorferi] 982 3.70E−130 f577.aagi|2688261 (AE001141) B. burgdorferi predicted 1930 4.00E−264 codingregion BB0352 [Borrelia f584.aa gi|2688246 (AE001140) B. burgdorferipredicted 1094 4.10E−147 coding region BB0346 [Borrelia f596.aagi|2688241 (AE001140) P26 [Borrelia burgdorferi] 13221.20E−180 >pir|G70141|G70141 P26 f596.aa gi|2281465 (AF000366) P26[Borrelia burgdorferi] 1010 5.90E−137 >gi|2281465 (AF000366) P26 f598.aagi|2281462 (AF000366) oligopeptide permease 652 1.20E−85 homolog D[Borrelia burgdorferi] f598.aa gi|143607 sporulation protein [Bacillussubtilis] 372 1.20E−45 f598.aa gnl|PID|e1183166 oligopeptide ABCtransporter (ATP- 372 1.20E−45 binding protein) [Bacillus f598.aagi|1574676 oligopeptide transport ATP-binding 344 6.70E−42 protein(oppD) [Haemophilus f598.aa gi|677943 AppD [Bacillus subtilis] 3448.00E−42 >gnl|PID|e1183156 oligopeptide ABC f598.aa gi|1787051(AE000185) o612; 48 pct identical (33 346 2.50E−41 gaps) to 525 residuesfrom f598.aa gi|47346 AmiE protein [Streptococcus 338 1.10E−40pneumoniae] >pir|S11152|S11152 amiE f598.aa gi|47805 Opp D (AA1-335)[Salmonella 332 5.70E−40 typhimurium] >sp|P04285|OPPD_SALTY f598.aapir|A03413|QREBOT oligopeptide transport protein oppD - 332 5.70E−40Salmonella typhimurium f598.aa gi|1787499 (AE000223) oligopeptidetransport 332 5.90E−40 ATP-binding protein OppD f598.aa gnl|PID|d1015494Oligopeptide transport ATP-binding 332 5.90E−40 protein OppD.[Escherichia f598.aa gi|495177 ATP binding protein [Lactococcus 3318.40E−40 lactis] >sp|P50980|OPPD_LACLC f598.aa gnl|PID|e187587oligopeptidepermease [Streptococcus 331 1.10E−39 pyogenes] f598.aagi|308850 ATP binding protein [Lactococcus 329 1.60E−39lactis] >pir|A53290|A53290 f598.aa gi|2313399 (AE000548) dipeptide ABCtransporter, 322 2.30E−39 ATP-binding protein (dppD) f6-21.aa gi|2281468(AF000948) OppAIV [Borrelia 565 4.30E−73 burgdorferi] >gi|2689891(AE000792) f6-21.aa gi|2253286 (AF005657) plasminogen binding 3151.20E−37 protein [Borrelia burgdorferi] f6-21.aa gi|2688228 (AE001139)oligopeptide ABC 314 1.60E−37 transporter, periplasmic f6-21.aagi|2809544 (AF043071) oligopeptide permease 314 1.60E−37 periplasmicbinding protein f6-21.aa gi|2281457 (AF000366) oligopeptide permease 3141.60E−37 homolog AI [Borrelia burgdorferi] f6-21.aa gi|2688227(AE001139) oligopeptide ABC 290 3.90E−34 transporter, periplasmicf6-21.aa gi|2281458 (AF000366) oligopeptide permease 290 3.90E−34homolog AII [Borrelia burgdorferi] f6-21.aa gi|2281455 (AF000365)oligopeptide permease 279 9.90E−34 homolog AV [Borrelia burgdorferi]f6-21.aa gi|2690261 (AE000790) oligopeptide ABC 282 5.30E−33transporter, periplasmic f6-21.aa gi|1616644 P30 [Borrelia burgdorferi]271 6.70E−32 f6-21.aa gi|2688226 (AE001139) oligopeptide ABC 2685.00E−31 transporter, periplasmic f6-21.aa gi|2281459 (AF000366)oligopeptide permease 268 5.00E−31 homolog AIII [Borrelia f6-21.aagi|2809546 (AF043071) oligopeptide permease 268 5.00E−31 periplasmicbinding protein f6-21.aa bbs|161785 60 kda antigen [Borrelia coriaceae,255 2.90E−30 C053, ATCC 4338, Peptide, 514 f6-21.aa gi|2983834(AE000740) transporter (extracellular 154 3.50E−14 solute bindingprotein family f6-27.aa gi|2689911 (AE000792) B. burgdorferi predicted1773 7.30E−240 coding region BBB09 [Borrelia f6-5.aa gi|2689905(AE000792) B. burgdorferi predicted 932 7.50E−126 coding region BBB27[Borrelia f600.aa gi|2281461 (AF000366) oligopeptide permease 7311.40E−100 homolog C [Borrelia burgdorferi] f600.aa gi|2688244 (AE001140)oligopeptide ABC 731 1.40E−100 transporter, permease protein (oppC-1)f600.aa gi|143606 sporulation protein [Bacillus subtilis] 3725.00E−48 >pir|C38447|C38447 f600.aa gi|40007 OppC gene product [Bacillussubtilis] 372 5.00E−48 >gnl|PID|e1183165 oligopeptide f600.aa gi|1574677oligopeptide transport system permease 372 7.30E−48 protein (oppC)C[Haemophilus f600.aa gi|47804 Opp C (AA1-301) [Salmonella 366 4.20E−47typhimurium] >pir|C29333|QREBOC f600.aa gnl|PID|d1015493 Oligopeptidetransport system permease 366 4.20E−47 protein OppC. f600.aagnl|PID|e1181495 (AJ002571) DppC [Bacillus subtilis] 2671.70E−42 >gnl|PID|e1183314 f600.aa gi|1732315 transport system permeasehomolog 335 5.30E−42 [Listeria monocytogenes] f600.aa gi|580851 dciAC[Bacillus subtilis] 258 1.50E−40 >sp|P26904|DPPC_BACSU DIPEPTIDETRANSPORT f600.aa gnl|PID|d1011164 oligopeptide transport systempermease 240 2.50E−39 protein [Synechocystis f600.aa gi|677947 AppC[Bacillus subtilis] 236 2.80E−37 >gnl|PID|e1183160 oligopeptide ABCf600.aa gi|1813497 dipeptide transporter protein dppC 281 1.20E−35[Bacillus firmus] f600.aa sp|Q10623|Y021_MYCTU PUTATIVE PEPTIDETRANSPORT 290 1.50E−35 PERMEASE PROTEIN CY373.01C. f600.aa gi|1532201BldKA [Streptomyces coelicolor] 291 1.60E−35 f603.aa gi|2281460(AF000366) oligopeptide permease 1522 5.80E−214 homolog B [Borreliaburgdorferi] f603.aa gi|1574678 dipeptide transport system permease 3921.30E−100 protein (dppB) [Haemophilus f603.aa gnl|PID|e1183164oligopeptide ABC transporter 374 3.40E−96 (permease) [Bacillus subtilis]f603.aa gi|580897 OppB gene product [Bacillus subtilis] 3736.60E−96 >pir|S15231|B38447 f603.aa gi|47803 Opp B (AA1-306) [Salmonella371 6.70E−96 typhimurium] >pir|B29333|QREBOB f603.aa gi|1787497(AE000223) oligopeptide transport 364 3.50E−95 system permease proteinOppB f603.aa gnl|PID|d1015492 Oligopeptide transport system permease 3573.50E−94 protein OppB. f603.aa gi|580850 dciAB [Bacillus subtilis] 3509.10E−90 >gnl|PID|e1181494 (AJ002571) DppB f603.aa gi|551726 sporulationprotein [Bacillus subtilis] 374 2.40E−87 >gi|143605 sporulation f603.aagi|349226 transmembrane protein [Escherichia 293 9.60E−79coli] >gi|466682 dppB f603.aa gi|1787053 (AE000185) o306; This 306 aaORF is 284 3.80E−77 46 pct identical (32 gaps) to f603.aa gi|972895 DppB[Haemophilus influenzae] 301 2.50E−76 >gi|1574114 dipeptide transportsystem f603.aa gi|2182646 (AE000098) Y4tP [Rhizobium sp. 294 9.10E−74NGR234] >sp|Q53191|Y4TP_RHISN f603.aa gi|2983140 (AE000692) transporter(OppBC 169 2.30E−73 family) [Aquifex aeolicus] f603.aa gi|677946 AppB[Bacillus subtilis] 218 8.70E−73 >gnl|PID|e1183159 oligopeptide ABCf604.aa gi|2281459 (AF000366) oligopeptide permease 2818 0 homolog AIII[Borrelia f604.aa gi|2809546 (AF043071) oligopeptide permease 2818 0periplasmic binding protein f604.aa gi|2688226 (AE001139) oligopeptideABC 2823 0 transporter, periplasmic f604.aa gi|2688227 (AE001139)oligopeptide ABC 1738 1.40E−234 transporter, periplasmic f604.aagi|2281458 (AF000366) oligopeptide permease 1731 1.30E−233 homolog AII[Borrelia burgdorferi] f604.aa gi|2281468 (AF000948) OppAIV [Borrelia1675 3.60E−229 burgdorferi] >gi|2689891 (AE000792) f604.aa gi|2688228(AE001139) oligopeptide ABC 718 1.60E−204 transporter, periplasmicf604.aa gi|2809544 (AF043071) oligopeptide permease 718 3.00E−204periplasmic binding protein f604.aa gi|2253286 (AF005657) plasminogenbinding 718 4.10E−204 protein [Borrelia burgdorferi] f604.aa gi|2281457(AF000366) oligopeptide permease 714 2.00E−203 homolog AI [Borreliaburgdorferi] f604.aa bbs|161785 60 kda antigen [Borrelia coriaceae, 7041.20E−190 C053, ATCC 4338, Peptide, 514 f604.aa gi|2281455 (AF000365)oligopeptide permease 1402 1.80E−188 homolog AV [Borrelia burgdorferi]f604.aa gi|2690261 (AE000790) oligopeptide ABC 1400 3.40E−188transporter, periplasmic f604.aa gi|1616644 P30 [Borrelia burgdorferi]858 4.90E−117 f604.aa gi|47802 Opp A (AA1-542) [Salmonella 296 9.00E−114typhimurium] >gi|47808 precursor f606.aa gi|2281458 (AF000366)oligopeptide permease 2762 0 homolog AII [Borrelia burgdorferi] f606.aagi|2688227 (AE001139) oligopeptide ABC 2774 0 transporter, periplasmicf606.aa gi|2281468 (AF000948) OppAIV [Borrelia 1817 6.50E−245burgdorferi] >gi|2689891 (AE000792) f606.aa gi|2809546 (AF043071)oligopeptide permease 1739 3.10E−234 periplasmic binding protein f606.aagi|2688226 (AE001139) oligopeptide ABC 1738 4.20E−234 transporter,periplasmic f606.aa gi|2281459 (AF000366) oligopeptide permease 17332.00E−233 homolog AIII [Borrelia f606.aa bbs|161785 60 kda antigen[Borrelia coriaceae, 762 1.70E−202 C053, ATCC 4338, Peptide, 514 f606.aagi|2281455 (AF000365) oligopeptide permease 1456 1.80E−195 homolog AV[Borrelia burgdorferi] f606.aa gi|2690261 (AE000790) oligopeptide ABC1454 3.30E−195 transporter, periplasmic f606.aa gi|2253286 (AF005657)plasminogen binding 751 2.00E−192 protein [Borrelia burgdorferi] f606.aagi|2688228 (AE001139) oligopeptide ABC 751 2.70E−192 transporter,periplasmic f606.aa gi|2809544 (AF043071) oligopeptide permease 7516.90E−192 periplasmic binding protein f606.aa gi|2281457 (AF000366)oligopeptide permease 748 2.40E−191 homolog AI [Borrelia burgdorferi]f606.aa gi|1616644 P30 [Borrelia burgdorferi] 1220 7.30E−163 f606.aagi|47802 Opp A (AA1-542) [Salmonella 285 7.80E−106typhimurium] >gi|47808 precursor f607.aa gi|2281457 (AF000366)oligopeptide permease 2694 0 homolog AI [Borrelia burgdorferi] f607.aagi|2253286 (AF005657) plasminogen binding 2706 0 protein [Borreliaburgdorferi] f607.aa gi|2809544 (AF043071) oligopeptide permease 2708 0periplasmic binding protein f607.aa gi|2688228 (AE001139) oligopeptideABC 2714 0 transporter, periplasmic f607.aa bbs|161785 60 kda antigen[Borrelia coriaceae, 1272 3.80E−242 C053, ATCC 4338, Peptide, 514f607.aa gi|2809546 (AF043071) oligopeptide permease 718 1.40E−204periplasmic binding protein f607.aa gi|2688226 (AE001139) oligopeptideABC 718 3.60E−204 transporter, periplasmic f607.aa gi|2281459 (AF000366)oligopeptide permease 713 1.70E−203 homolog AIII [Borrelia f607.aagi|2688227 (AE001139) oligopeptide ABC 751 2.40E−192 transporter,periplasmic f607.aa gi|2281458 (AF000366) oligopeptide permease 7514.50E−192 homolog AII [Borrelia burgdorferi] f607.aa gi|2281468(AF000948) OppAIV [Borrelia 806 8.40E−189 burgdorferi] >gi|2689891(AE000792) f607.aa gi|2690261 (AE000790) oligopeptide ABC 601 1.20E−144transporter, periplasmic f607.aa gi|2281455 (AF000365) oligopeptidepermease 600 1.60E−144 homolog AV [Borrelia burgdorferi] f607.aagi|1616644 P30 [Borrelia burgdorferi] 709 5.40E−103 f607.aa gi|47802 OppA (AA1-542) [Salmonella 261 8.50E−69 typhimurium] >gi|47808 precursorf611.aa gi|2688231 (AE001139) B. burgdorferi predicted 1907 1.10E−261coding region BB0325 [Borrelia f617.aa gi|2688213 (AE001138) conservedhypothetical 1574 2.70E−226 integral membrane protein f617.aa gi|2649711(AE001042) ribose ABC transporter, 109 7.00E−12 permease protein(rbsC-1) f631.aa gi|1165286 FtsW [Borrelia burgdorferi] 18204.00E−259 >gi|2688164 (AE001137) cell division f631.aa gnl|PID|e229592membrane protein [Borrelia 1815 2.10E−258 burgdorferi] >gnl|PID|e228289ftsW f631.aa gi|146039 cell division protein [Escherichia coli] 3621.30E−60 >gi|40857 FtsW protein f631.aa gi|580938 internal open readingframe (AA 1-290) 407 4.90E−55 [Bacillus subtilis] f631.aagnl|PID|e315953 FtsW [Mycobacterium tuberculosis] 4125.40E−55 >sp|O06223|FTWH_MYCTU f631.aa gi|580937 spoVE gene product (AA1-366) 410 2.90E−53 [Bacillus subtilis] >gnl|PID|e1185111 f631.aagi|143657 endospore forming protein [Bacillus 405 1.20E−52 subtilis]f631.aa gnl|PID|d1019002 rod-shape-determining protein 358 3.10E−51[Synechocystis sp. ] f631.aa gnl|PID|e1287793 (AL022602) cell divisinprotein FtsW 396 6.70E−51 [Mycobacterium leprae] f631.aa gi|1016213strong sequence similarity to FtsW, 349 1.00E−50 RodA, and SpoV-E[Cyanophora f631.aa gi|1574692 cell division protein (ftsW) 304 4.20E−50[Haemophilus influenzae] f631.aa gnl|PID|e1185075 similar tocell-division protein [Bacillus 281 1.80E−46 subtilis] f631.aagi|1469784 putative cell division protein ftsW 247 1.60E−38[Enterococcus hirae] f631.aa gi|1572976 rod shape-determining protein(mreB) 196 1.20E−37 [Haemophilus influenzae] f631.aa gi|147695rod-shape-determining protein 194 5.00E−35 [Escherichiacoli] >gi|1778551 f635.aa gi|1165282 orf7; Method: conceptualtranslation 1166 1.00E−156 supplied by author [Borrelia f635.aagi|1448949 ORF 224; The predicted gene product 621 2.80E−125 showed weakhomology with the f647.aa gi|2688180 (AE001137) flagellar protein (flbB)1032 1.00E−140 [Borrelia burgdorferi] f647.aa gi|1196323 putative[Borrelia burgdorferi] 1031 1.50E−140 f647.aa gi|1165270 orf19; Method:conceptual translation 1019 7.10E−139 supplied by author [Borreliaf647.aa gi|2108242 22.5 K protein [Treponema pallidum] 200 4.70E−24f65.aa gi|2688737 (AE001178) B. burgdorferi predicted 1095 8.10E−148coding region BB0792 [Borrelia f653.aa gi|1165265 MotB [Borreliaburgdorferi] 1220 1.70E−164 >gi|1185054 flagellar motor apparatusf653.aa gi|1399286 MotB [Treponema phagedenis] 168 5.80E−57 f653.aagi|2196896 MotB [Treponema pallidum] 179 1.30E−49 f664.aa gi|1185062flagellar export protein [Borrelia 1430 1.90E−199 burgdorferi] f664.aagi|1165257 FlhB [Borrelia burgdorferi] 1430 1.90E−199 >gi|2688194(AE001137) flagellar f664.aa gi|1216382 FlhB′ [Treponema pallidum] 2725.30E−64 >pir|PC4115|PC4115 flagellar protein f664.aa gi|395390flagellar biosynthetic protein [Bacillus 433 1.30E−61 subtilis] f664.aagnl|PID|e1185229 flagella-associated protein [Bacillus 433 1.30E−61subtilis] f664.aa gi|1147737 third gene in fliQ operon; membrane 3531.70E−46 protein homolog [Caulobacter f664.aa gi|2313898 (AE000589)flagellar biosynthetic 203 1.20E−44 protein (flhB) [Helicobacter f664.aagi|2984250 (AE000768) flagellar biosynthetic 319 3.00E−44 protein FlhB[Aquifex aeolicus] f664.aa gi|2459702 FlhB [Agrobacterium tumefaciens]347 6.20E−44 f664.aa gi|793892 flhB [Yersinia enterocolitica] 3301.30E−39 >pir|S54213|S54213 flhB protein - f664.aa gnl|PID|d1016420Flagellar biosynthetic protein FlhB. 325 2.20E−39 [Escherichia coli]f664.aa gi|475126 yscU [Yersinia pseudotuberculosis] 3099.80E−38 >gi|2996233 (AF053946) Yop f664.aa gi|497216 YscU [Yersiniaenterocolitica] 308 1.40E−37 f664.aa gnl|PID|d1007477 flagellar proteinFlhB [Salmonella 312 2.10E−37 typhimurium] f664.aa gnl|PID|e283684secretion system apparatus, SsaU 312 8.20E−37 [Salmonella typhimurium]f679.aa gi|2688158 (AE001136) B. burgdorferi predicted 3714 0 codingregion BB0259 [Borrelia f679.aa gnl|PID|d1011473 soluble lytictransglycosylase 180 1.10E−25 [Synechocystis sp. ] f679.aagnl|PID|e1183177 similar to lytic transglycosylase 108 2.10E−22[Bacillus subtilis] f679.aa gi|2984090 (AE000756) hypothetical protein111 9.30E−17 [Aquifex aeolicus] f680.aa gi|2688153 (AE001136) bacitracinresistance 769 3.90E−109 protein (bacA) [Borrelia f680.aagnl|PID|e1185988 similar to bacitracin resistance protein 174 7.30E−18(undecaprenol f680.aa gi|2622542 (AE000905) bacitracin resistance 1163.30E−16 protein [Methanobacterium f680.aa gi|2984378 (AE000777)undecaprenol kinase 152 3.90E−15 [Aquifex aeolicus] f680.aa gi|882579 CGSite No. 29739 [Escherichia coli] 139 2.60E−12 >gi|1789437 (AE000387)f688.aa gi|2688146 (AE001135) conserved hypothetical 2497 0 integralmembrane protein f688.aa gi|2649351 (AE001019) conserved hypothetical110 3.70E−18 protein [Archaeoglobus fulgidus] f688.aa gi|1592186 M.jannaschii predicted coding region 174 1.10E−16 MJ1562 [Methanococcusf7-30.aa gi|2690009 (AE000786) conserved hypothetical 682 1.90E−90protein [Borrelia burgdorferi] f704.aa gi|2688137 (AE001134) glyceroluptake facilitator 1307 4.70E−181 (glpF) [Borrelia f704.aa gi|142997glycerol uptake facilitator [Bacillus 191 1.50E−50subtilis] >gnl|PID|e1182917 f704.aa gi|521003 C01G6.1 [Caenorhabditiselegans] 152 1.60E−50 f704.aa gi|529582 water channel protein [Rattus142 5.80E−50 norvegicus] >pir|I59266|I59266 water f704.aadbjl|AB000507_1 (AB000507) aquaporin 7 [Rattus 155 1.30E−49 norvegicus]f704.aa pir|A57119|A57119 aquaporin 3 - human 149 4.20E−44 f704.aagi|1109920 coded for by C. elegans cDNA 168 9.30E−44 cm16b11; strongsimilarity to MIP f704.aa gnl|PID|d1019987 (AB001325) aquaporin 3 [Homo148 5.30E−43 sapiens] >sp|Q92482|AQP3_HUMAN f704.aa gnl|PID|d1025786(AB008775) aquaporin 9 [Homo 144 1.40E−42 sapiens] f704.aa gi|146188glycerol diffusion facilitator 146 1.30E−40 [Escherichiacoli] >gi|305030 CG Site f704.aa gi|1065485 strong similarity to the MIPfamily of 179 1.40E−39 transmembrane channel f704.aasp|P31140|GLPF_SHIFL GLYCEROL UPTAKE 146 3.30E−39 FACILITATOR PROTEIN.f704.aa gi|2587035 (AF026270) PduF [Salmonella 168 7.30E−39typhimurium] >sp|P37451|PDUF_SALTY f704.aa gi|1399489 glyceroldfiffusion facilitator 154 7.90E−39 [Pseudomonas aeruginosa] f704.aagi|2649144 (AE001005) glycerol uptake facilitator, 150 1.30E−38 MIPchannel (glpF) f707.aa gi|2688143 (AE001134) B. burgdorferi predicted1300 3.90E−176 coding region BB0238 [Borrelia f709.aa gi|2688131(AE001133) B. burgdorferi predicted 3437 0 coding region BB0236[Borrelia f730.aa gi|2688111 (AE001132) gufA protein [Borrelia 13763.00E−192 burgdorferi] >pir|C70127|C70127 f730.aa gi|1707057 coded forby C. elegans cDNA 235 2.80E−83 CEESS55F; coded for by C. elegans cDNAf730.aa gi|2621542 (AE000831) conserved protein 259 1.10E−74[Methanobacterium thermoautotrophicum] f730.aa gnl|PID|e183440 gufA geneproduct [Myxococcus 175 2.30E−35 xanthus] >gi|49253 orfX gene f730.aagi|2984109 (AE000757) hypothetical protein 171 7.00E−28 [Aquifexaeolicus] f736.aa gi|2688115 (AE001132) phosphate ABC 1403 2.10E−186transporter, periplasmic phosphate- binding f736.aa gi|2622858(AE000929) phosphate-binding protein 151 4.40E−30 PstS [Methanobacteriumf736.aa gi|2622859 (AE000929) phosphate-binding protein 145 2.80E−24PstS homolog [Methanobacterium f736.aa gnl|PID|d1010224 ORF108 [Bacillussubtilis] 120 1.20E−11 >gnl|PID|e1185766 alternate gene f739.aagi|2688119 (AE001132) B. burgdorferi predicted 1139 1.10E−156 codingregion BB0213 [Borrelia f742.aa gi|2688100 (AE001131) surface-locatedmembrane 5654 0 protein 1 (lmp1) [Borrelia f742.aa gi|2621120 (AE000799)O-linked GlcNAc 200 9.30E−22 transferase [Methanobacterium f742.aagi|2621106 (AE000798) O-linked GlcNAc 180 5.80E−17 transferase[Methanobacterium f742.aa pir|E69190|E69190 conserved hypotheticalprotein MTH68 - 154 1.60E−14 Methanobacterium f742.aa gi|1591608transformation sensitive protein 109 9.90E−14 [Methanococcus jannaschii]f742.aa gi|1589778 SPINDLY [Arabidopsis thaliana] 101 1.40E−13 f742.aagi|2984175 (AE000762) hypothetical protein 132 7.30E−13 [Aquifexaeolicus] f742.aa gi|3037137 (AF056198) Hsp70/Hsp90 organizing 1055.40E−11 protein homolog [Drosophila f743.aa gi|2688104 (AE001131) B.burgdorferi predicted 1299 1.70E−174 coding region BB0209 [Borreliaf748.aa gi|2688089 (AE001130) Lambda CII stability- 1615 5.10E−220governing protein (hflC) [Borrelia f748.aa gi|436158 putative integralmembrane protease 191 4.80E−35 required for high frequency f748.aagi|1573107 Lambda CII stability-governing protein 193 4.90E−33 (hflC)[Haemophilus f748.aa gi|507735 HflC [Vibrio parahaemolyticus] 2126.10E−26 >sp|P40606|HFLC_VIBPA HFLC PROTEIN f752.aa gi|2688092(AE001130) 2585 0 f752.aa gi|2984050 (AE000754) UDP-MurNac-tripeptide202 9.10E−74 synthetase [Aquifex aeolicus] f752.aa gi|40162 murE geneproduct [Bacillus subtilis] 157 6.40E−70 >gnl|PID|e1185108 f752.aagnl|PID|d1011466 UDP-MurNac-tripeptide synthetase 166 5.20E−57[Synechocystis sp. ] f752.aa gnl|PID|e307808 UDP-MurNAc-tripeptidesynthetase 108 2.30E−51 [Rickettsia prowazekii] f752.aa gi|1574688UDP-MurNac-tripeptide synthetase 166 3.20E−50 (murE) [Haemophilusinfluenzae] f752.aa gnl|PID|e1287797 (AL022602) udp-n- 183 3.20E−50acetylmuramoylalanyl-d-glutamate f752.aa gnl|PID|e316022 MurE[Mycobacterium tuberculosis] 181 4.10E−46 f752.aa gi|581032UDP-MurNac-tripeptide synthetase 175 1.30E−41 (MurE) [Escherichia coli]f752.aa gi|2177098 UDP-MurNAc-Dipeptide: meso- 172 3.70E−41diaminopimelate ligase [Escherichia f752.aa gi|2314673 (AE000648)UDP-MurNac-tripeptide 137 9.80E−41 synthetase (murE) [Helicobacterf752.aa gi|840843 UDP-N-acetylmuramoylalanyl-D- 135 1.70E−20glutamate-2,6-diaminopimelate ligase f76-1.aa gi|1209837 lipoprotein[Borrelia burgdorferi] 395 2.80E−49 f76-1.aa gi|1209873 lipoprotein[Borrelia burgdorferi] 250 7.00E−37 f76-1.aa gi|1209843 lipoprotein[Borrelia burgdorferi] 267 7.30E−32 f76-1.aa gi|2121280 (AF000270)lipoprotein [Borrelia 258 1.20E−30 burgdorferi] >gi|3095109 f76-1.aagnl|PID|e268244 surface-exposed lipoprotein [Borrelia 116 2.40E−18afzelii] f76-1.aa gi|1209849 lipoprotein [Borrelia burgdorferi] 1468.30E−17 f76-1.aa gi|3095105 (AF046998) 2.9-8 lipoprotein [Borrelia 1485.80E−14 burgdorferi] f76-1.aa gi|3095107 (AF046999) 2.9-9 lipoprotein[Borrelia 127 7.20E−11 burgdorferi] f764.aa gi|2688084 (AE001129) B.burgdorferi predicted 1218 1.20E−164 coding region BB0193 [Borreliaf770.aa gi|2688077 (AE001129) conserved hypothetical 646 7.60E−87protein [Borrelia burgdorferi] f790.aa gi|2688065 (AE001128) outermembrane protein 2013 2.50E−271 (tpn50) [Borrelia burgdorferi] f790.aagi|458015 TpN50 precursor [Treponema pallidum] 134 4.30E−33 f790.aasp|P38369|TP50_TREPA OUTER MEMBRANE PROTEIN 134 4.30E−33 TPN50PRECURSOR. f790.aa gi|532658 antigen [Treponema pallidum] 1394.30E−31 >pir|S61867|S61867 antigen tpp57 - f792.aa gi|2688052(AE001127) B. burgdorferi predicted 3185 0 coding region BB0165[Borrelia f797.aa gi|2688056 (AE001127) B. burgdorferi predicted 11165.30E−148 coding region BB0159 [Borrelia f798.aa gi|2688051 (AE001127)antigen, S2, putative 1223 9.70E−164 [Borrelia burgdorferi] f798.aagi|1063419 S2 gene product [Borrelia burgdorferi] 116 4.70E−23 f798.aagi|2690227 (AE000790) antigen, S2 [Borrelia 116 1.50E−22burgdorferi] >pir|D70207|D70207 f798.aa gi|2690128 (AE000788) proteinp23 [Borrelia 110 1.40E−19 burgdorferi] >pir|C70257|C70257 f798.aagi|2689956 (AE000785) protein p23 [Borrelia 104 2.70E−15burgdorferi] >pir|D70225|D70225 f799.aa gi|2688043 (AE001126) B.burgdorferi predicted 632 1.40E−83 coding region BB0156 [Borreliaf8-10.aa gi|2690052 (AE000784) antigen, P35, putative 1241 1.10E−167[Borrelia burgdorferi] f8-10.aa gi|2689955 (AE000785) antigen, P35,putative 298 1.70E−57 [Borrelia burgdorferi] f8-10.aa gi|2690120(AE000789) B. burgdorferi predicted 254 3.80E−54 coding region BBI34[Borrelia f8-10.aa gi|2690100 (AE000789) B. burgdorferi predicted 1822.90E−31 coding region BBI16 [Borrelia f8-10.aa gi|2690207 (AE000787) B.burgdorferi predicted 196 1.50E−20 coding region BBJ02 [Borreliaf8-10.aa gi|2690116 (AE000789) B. burgdorferi predicted 192 5.50E−20coding region BBI29 [Borrelia f8-10.aa gi|2690125 (AE000788) antigen,P35, putative 129 5.80E−14 [Borrelia burgdorferi] f8-10.aa gi|2690206(AE000787) B. burgdorferi predicted 103 1.10E−13 coding region BBJ01[Borrelia f8-10.aa gi|2690099 (AE000789) B. burgdorferi predicted 1428.50E−13 coding region BBI15 [Borrelia f8-10.aa gi|2690115 (AE000789) B.burgdorferi predicted 130 3.30E−12 coding region BBI28 [Borreliaf8-14.aa gi|2690074 (AE000784) B. burgdorferi predicted 1560 2.60E−206coding region BBH37 [Borrelia f8-14.aa gi|2690188 (AE000787) B.burgdorferi predicted 599 3.50E−123 coding region BBJ08 [Borreliaf8-14.aa gi|2690030 (AE000786) B. burgdorferi predicted 337 4.40E−106coding region BBG01 [Borrelia f8-14.aa gi|2690139 (AE000788) B.burgdorferi predicted 173 8.00E−91 coding region BBK01 [Borrelia f8.aagi|2688783 (AE001182) B. burgdorferi predicted 2765 0 coding regionBB0840 [Borrelia f8.aa gi|2697112 (AF008219) unknown [Borrelia afzelii]1494 2.80E−205 f800.aa gi|2688044 (AE001126) B. burgdorferi predicted1936 1.00E−262 coding region BB0155 [Borrelia f805.aa gi|2688039(AE001126) N-acetylglucosamine-6- 641 6.30E−85 phosphate deacetylase(nagA) f810.aa gi|2688024 (AE001125) glycine betaine, L-proline 15274.20E−207 ABC transporter, f810.aa gi|984805 glycine betaine-bindingprotein 179 6.80E−21 precursor [Bacillus subtilis] f810.aa gi|1850605ProX [Streptococcus mutans] 181 2.30E−18 f814.aa pir|D70117|D70117acriflavine resistance protein (acrB) 5105 0 homolog - Lyme diseasef814.aa gi|2688027 (AE001125) acriflavine resistance 5111 0 protein(acrB) [Borrelia f814.aa gi|2983346 (AE000707) cation efflux 3254.80E−119 (AcrB/AcrD/AcrF family) [Aquifex aeolicus] f814.aa gi|2313726(AE000574) acriflavine resistance 327 4.50E−111 protein (acrB)[Helicobacter f814.aa gi|3068786 (AF059041) RND pump protein 2971.70E−110 [Helicobacter pylori] f814.aa gnl|PID|e1182651 similar toacriflavin resistance protein 257 8.90E−100 [Bacillus subtilis] f814.aagi|1573914 acriflavine resistance protein (acrB) 294 2.10E−97[Haemophilus influenzae] f814.aa gnl|PID|e256815 mexF [Pseudomonasaeruginosa] 300 2.00E−88 f814.aa gnl|PID|d1019295 cation efflux systemprotein CzcA 198 1.30E−87 [Synechocystis sp. ] f814.aa gnl|PID|e285274membrane-bound cation-proton- 283 2.20E−87 antiporter [Ralstoniaeutropha] f814.aa gi|438854 envD homologue; ORFB 290 6.50E−87[Pseudomonas aeruginosa] >pir|S39630|S39630 f814.aa gnl|PID|d1011721CzcA [Alcaligenes sp. ] 275 8.20E−87 >pir|JC4700|JC4700 cadmium, zinc,f814.aa gi|2314107 (AE000605) cation efflux system 266 2.30E−86 protein(czcA) [Helicobacter f814.aa pir|A33830|A33830 cation efflux systemmembrane protein 275 3.10E−86 czcA - Alcaligenes f814.aagnl|PID|d1017073 envD gene product homolog 283 8.30E−86 [Escherichiacoli] >gi|1788814 f818.aa gi|2688032 (AE001125) B. burgdorferi predicted664 3.00E−87 coding region BB0139 [Borrelia f82.aa gi|2688729 (AE001177)B. burgdorferi predicted 991 2.20E−132 coding region BB0776 [Borreliaf820.aa gi|2688029 (AE001125) penicillin-binding protein 3171 0 (pbp-1)[Borrelia f820.aa gi|580936 SpoVD [Bacillus subtilis] 1493.00E−49 >gnl|PID|e1185107 penicillin-binding f820.aa gi|150283penicillin-binding protein 2 [Neisseria 154 6.90E−43 meningitidis]f820.aa gnl|PID|e1287798 (AL022602) penicillin binding protein 2 1824.20E−42 [Mycobacterium f820.aa gi|509190 penicillin-binding protein 2[Neisseria 158 1.70E−41 meningitidis] f820.aa gi|509118penicillin-binding protein 2 [Neisseria 151 7.10E−41 meningitidis]f820.aa gi|840842 penicillin-binding protein 3 177 1.20E−40 [Pseudomonasaeruginosa] f820.aa gi|509065 penicillin-binding protein 2 [Neisseria152 1.40E−40 meningitidis] f820.aa gi|509043 penicillin-binding protein2 [Neisseria 150 2.70E−40 meningitidis] f820.aa gi|509159penicillin-binding protein 2 [Neisseria 147 2.80E−40 meningitidis]f820.aa gi|509120 penicillin-binding protein 2 [Neisseria 155 1.60E−39meningitidis] f820.aa gi|509157 penicillin-binding protein 2 [Neisseria155 1.60E−39 meningitidis] f820.aa gi|509126 penicillin-binding protein2 [Neisseria 158 1.70E−39 meningitidis] f820.aa gi|45178penicillin-binding protein 2 (AA 1- 155 2.30E−38 581) [Neisseriameningitidis] f820.aa gi|150279 penicillin binding protein 2 [Neisseria154 8.70E−38 gonorrhoeae] f831.aa gi|2688018 (AE001124) B. burgdorferipredicted 994 1.20E−133 coding region BB0126 [Borrelia f843.aagi|2688014 (AE001124) PTS system, maltose and 2590 0 glucose-specificIIABC component f843.aa gi|2688579 (AE001166) PTS system, glucose- 5941.80E−129 specific IIBC component (ptsG) f843.aa gi|1072418 glcA[Staphylococcus carnosus] 283 1.00E−72 >pir|S46952|S46952 f843.aagi|1072419 glcB [Staphylococcus carnosus] 2481.00E−66 >pir|S63606|S46953 f843.aa dbj∥D86417_11 YflF [Bacillussubtilis] 215 7.90E−65 >gnl|PID|e1182760 similar to f843.aa gi|2197104(AF003742) MalX homolog 182 8.90E−64 [Escherichia coli] f843.aa gi|43819nagE gene product [Klebsiella 264 8.50E−63pneumoniae] >pir|S18607|S18607 f843.aa gi|146913 N-acetylglucosaminetransport protein 256 1.10E−62 [Escherichia coli] f843.aa gi|39956 IIGlc[Bacillus subtilis] 315 5.20E−62 >gnl|PID|e1184979 phosphotransferasesystem f843.aa dbj∥D87820_1 NagE [Vibrio cholerae non-O1] 2633.80E−61 >pir|JC5651|JC5651 f843.aa gi|2689888 (AE000792) PTS system,maltose and 198 1.10E−60 glucose-specific IIABC component f843.aagi|397363 enzyme II-glc [Salmonella 227 1.20E−58typhimurium] >pir|S36620|S36620 f843.aa gi|147393 glucose-specificenzyme II of 226 3.90E−57 phosphotransferase system [Escherichia f843.aagnl|PID|e1182187 alternate gene name: yzfA; similar to 180 9.00E−56phosphotransferase f843.aa gi|1732194 PTS permease for glucose [Vibrio349 4.30E−50 furnissii] f850.aa gi|2687999 (AE001123) B. burgdorferipredicted 2374 0 coding region BB0110 [Borrelia f853.aa gi|2687994(AE001123) basic membrane protein 1672 2.20E−224 [Borrelia burgdorferi]f853.aa gi|155055 basic membrane protein precursor 130 3.60E−24[Treponema pallidum] f859.aa gi|2688002 (AE001123) B. burgdorferipredicted 888 1.80E−115 coding region BB0102 [Borrelia f86.aa gi|2688725(AE001177) flagellar P-ring protein 1647 1.50E−217 (flgI) [Borreliaburgdorferi] f86.aa gi|2920802 (AF019213) FlgI [Vibrio cholerae] 1433.50E−14 f86.aa gi|405550 flagellar P-ring protein [Pseudomonas 1023.70E−13 putida] >sp|Q52082|FLGI_PSEPU f86.aa gi|144241 flagellin[Caulobacter crescentus] 110 6.70E−13 >pir|A41891|A41891 basal bodyf860.aa gi|2687998 (AE001123) asparaginyl-tRNA 1110 2.40E−149 synthetase(asnS) [Borrelia f860.aa gi|1574761 asparaginyl-tRNA synthetase (asnS)634 1.30E−83 [Haemophilus influenzae] f860.aa gi|147935 asparaginyl-tRNAsynthetase (asnS) 622 6.10E−82 [Escherichia coli] >gi|41000 f860.aagnl|PID|e1202698 (AJ222644) asparaginyl-tRNA 404 2.40E−80 synthetase[Arabidopsis thaliana] f860.aa gnl|PID|d1011495 asparaginyl-tRNAsynthetase 618 4.50E−80 [Synechocystis sp. ] f860.aa gi|530408 Asn-tRNAsynthetase [Mycoplasma 439 1.60E−65 capricolum] >pir|S77842|S77842f860.aa gi|1045792 asparaginyl-tRNA synthetase 365 2.20E−62 [Mycoplasmagenitalium] f860.aa gi|1674281 (AE000057) Mycoplasma pneumoniae, 3383.10E−61 asparaginyl-tRNA synthetase; f860.aa gnl|PID|e1202700(AJ222645) asparaginyl-tRNA 364 3.90E−59 synthetase [Arabidopsisthaliana] f860.aa gnl|PID|e264488 YCR024c, len: 492 [Saccharomyces 1503.90E−47 cerevisiae] >pir|S19435|S19435 f860.aa gnl|PID|e254305asparaginyl-tRNA synthetase 370 1.70E−46 [Salmonella typhi] f860.aagnl|PID|e188505 asparagine-tRNA ligase [Lactobacillus 224 1.30E−44delbrueckii] f860.aa pir|S71072|S71072 asparagine-tRNA ligase (EC6.1.1.22) 224 1.30E−44 asnS1 - Lactobacillus f860.aa gnl|PID|e188572asparagine-tRNA ligase [Lactobacillus 224 2.40E−44 delbrueckii] f860.aagi|1146247 asparaginyl-tRNA synthetase [Bacillus 234 6.10E−44subtilis] >gnl|PID|e1183681 f861.aa gi|2687975 (AE001122) glutamateracemase (murI) 1354 2.90E−186 [Borrelia burgdorferi] f861.aa gi|396314glutamate synthase [Escherichia coli] 168 1.20E−16 >gi|290428 glutamatesynthase f861.aa gnl|PID|e1165353 glutamate racemase [Bacillus subtilis]120 1.80E−13 >gnl|PID|e1184088 f861.aa pir|JC5587|JC5587 glutamateracemase (EC 5.1.1.3) - 122 1.80E−13 Bacillus pumilus f861.aasp|P52973|MURI_HAEIN PROBABLE GLUTAMATE 114 8.10E−13 RACEMASE (EC5.1.1.3). f867.aa gi|2687979 (AE001122) V-type ATPase, subunit A 2826 0(atpA) [Borrelia burgdorferi] f867.aa pir|JC5532|JC5532 vacuolar-typeATPase (EC 3.-.-.-) A 594 2.20E−162 chain -Desulfurococcus f867.aagi|2104726 V-ATPase A subunit [Desulfurococcus 594 3.10E−162 sp. SY]f867.aa gi|2605627 ATPase alpha subunit [Thermococcus 592 7.10E−161 sp.]f867.aa gnl|PID|d1003475 Na+-ATPase alpha subunit 601 1.60E−153[Enterococcus hirae] f867.aa gi|1590955 H+-transporting ATP synthase,subunit 585 6.00E−147 A (atpA) [Methanococcus f867.aa gi|496904 membraneATPase [Haloferax volcanii] 728 6.00E−147 >pir|S55895|S45144 f867.aagi|152927 ATPase alpha subunit [Sulfolobus 548 5.00E−163acidocaldarius] >pir|A28652|A28652 f867.aa gi|2649416 (AE001023)H+-transporting ATP 748 2.00E−146 synthase, subunit A (atpA) f867.aagi|2622052 (AE000869) ATP synthase, subunit A 607 9.40E−146[Methanobacterium f867.aa gi|168926 vacuolar ATPase vma-1 [Neurospora302 9.00E−145 crassa] >pir|A30799|PXNCV7 f867.aa gi|149820 ATPase alphasubunit [Methanosarcina 743 1.40E−143 barkeri] >pir|A34283|A34283f867.aa gi|160736 vacuolar ATPase [Plasmodium 305 9.40E−140falciparum] >pir|A48582|A48582 vacuolar f867.aa gnl|PID|d1009732adenosine triphosphatase A subunit 307 9.00E−137 [Acetabulariaacetabulum] f867.aa gi|49048 ATPase alpha-subunit [Thermus 684 4.80E−136aquaticus thermophilus] f868.aa gi|2687980 (AE001122) V-type ATPase,subunit B 2205 1.80E−298 (atpB) [Borrelia burgdorferi] f868.aagi|1590954 H+-transporting ATP synthase, subunit 156 2.00E−114 B (atpB)[Methanococcus f868.aa gi|2605628 ATPase beta subunit [Thermococcus 1513.30E−108 sp.] f868.aa gi|2104727 V-ATPase B subunit [Desulfurococcus151 1.10E−107 sp. SY] f868.aa gi|43641 ATP synthase subunit[Halobacterium 150 1.80E−107 salinarium] >pir|S14733|S14733 f868.aagi|149821 ATPase beta subunit [Methanosarcina 172 1.00E−105barkeri] >pir|B34283|B34283 f868.aa gnl|PID|d1003476 Na+-ATPase betasubunit 151 1.40E−105 [Enterococcus hirae] f868.aa gi|2649415 (AE001023)H+-transporting ATP 151 1.70E−103 synthase, subunit B (atpB) f868.aagi|496905 membrane ATPase [Haloferax volcanii] 1535.80E−103 >pir|S55896|S45145 f868.aa gi|1199639 A1AO H+ ATPase, subunitB 173 2.20E−102 [Methanosarcina mazeii] f868.aa gi|2622051 (AE000869)ATP synthase, subunit B 155 1.00E−101 [Methanobacterium f868.aagnl|PID|d1009734 adenosine triphosphatase B subunit 159 1.30E−101[Acetabularia acetabulum] f868.aa gi|1086645 Similar to vacuolar ATPsynthase 163 1.30E−101 (strong). [Caenorhabditis elegans] f868.aagi|459198 vacuolar H+- ATPase subunit B 164 4.60E−101 [Gossypiumhirsutum] f868.aa gi|167108 vacuolar ATPase B subunit [Hordeum 1644.60E−101 vulgare] >sp|Q40078|VAT1_HORVU f872.aa gi|2687986 (AE001122)B. burgdorferi predicted 1684 1.60E−230 coding region BB0089 [Borreliaf874.aa gi|2687965 (AE001121) L-lactate dehydrogenase 1603 2.80E−217(ldh) [Borrelia burgdorferi] f874.aa gi|39758 L-lactate dehydrogenase[Bacillus 520 3.10E−109 psychrosaccharolyticus] f874.aapir|S08183|S08183 L-lactate dehydrogenase (EC 1.1.1.27) 515 4.30E−109X - Bacillus f874.aa pir|A25805|A25805 L-lactate dehydrogenase (EC1.1.1.27) - 520 1.00E−107 Bacillus subtilis f874.aa gi|143136 L-lactatedehydrogenase [Bacillus 430 5.20E−107 megaterium] >pir|S00133|DEBSLMf874.aa gi|143138 lactate dehydrogenase (EC 1.1.1.27) 514 6.60E−107[Bacillus stearothermophilus] f874.aa gnl|PID|d1009574 L-lactatedehydrogenase [Bacillus 512 8.90E−107 subtilis] >gnl|PID|e1182257f874.aa gi|143134 lactate dehydrogenase (EC 1.1.1.27) 516 1.70E−106[Bacillus caldotenax] f874.aa gi|143132 lactate dehydrogenase (AC1.1.1.27) 506 2.30E−106 [Bacillus caldolyticus] f874.aa gi|412392NAD-dependent dehydrogenase 508 4.40E−106 [unidentified] f874.aagi|143130 L-lactate dehydrogenase [Bacillus 510 1.10E−105caldotenax] >pir|S00019|S00019 f874.aa gi|642256 L-lactate dehydrogenase[Pediococcus 560 1.70E−91 acidilactici] f874.aa gi|847956 L-lactatedehydrogenase [Lactobacillus 381 2.30E−91 sake] >sp|P50934|LDH_LACSKf874.aa gi|581305 L-lactate dehydrogenase [Lactobacillus 547 2.30E−91plantarum] >pir|A36957|A36957 f874.aa gi|149575 L(+)-lactatedehydrogenase 386 3.20E−91 [Lactobacillus casei] f886.aa gi|2687958(AE001120) B. burgdorferi predicted 1792 9.50E−237 coding region BB0077[Borrelia f888.aa gi|2687959 (AE001120) B. burgdorferi predicted 23513.59999944710933e−318 coding region BB0075 [Borrelia f893.aa gi|2687962(AE001120) B. burgdorferi predicted 2514 0 coding region BB0071[Borrelia f895.aa gi|2687954 (AE001120) conserved hypothetical 7473.60E−100 protein [Borrelia burgdorferi] f895.aa gnl|PID|e1184285similar to hypothetical proteins 103 2.50E−35 [Bacillus subtilis]f899.aa gi|2687946 (AE001119) B. burgdorferi predicted 1161 4.30E−158coding region BB0066 [Borrelia f924.aa gi|2687934 (AE001118) B.burgdorferi predicted 692 3.90E−93 coding region BB0044 [Borreliaf925.aa gi|2687935 (AE001118) B. burgdorferi predicted 1771 7.50E−242coding region BB0043 [Borrelia f929.aa gi|2687916 (AE001117) B.burgdorferi predicted 2589 0 coding region BB0038 [Borrelia f93.aagi|2688703 (AE001176) pyridoxal kinase (pdxK) 1334 6.60E−181 [Borreliaburgdorferi] f933.aa gi|2687917 (AE001117) B. burgdorferi predicted 9021.90E−122 coding region BB0034 [Borrelia f933.aa gi|2690091 (AE000789)conserved hypothetical 136 3.10E−37 protein [Borrelia burgdorferi]f933.aa gi|2690225 (AE000790) conserved hypothetical 149 4.50E−37protein [Borrelia burgdorferi] f933.aa gi|2690045 (AE000784) conservedhypothetical 126 5.70E−28 protein [Borrelia burgdorferi] f933.aagi|2239281 No definition line found [Borrelia 148 2.40E−14 burgdorferi]f939.aa gi|2687919 (AE001117) B. burgdorferi predicted 1796 7.50E−241coding region BB0028 [Borrelia f940.aa gi|2687920 (AE001117) B.burgdorferi predicted 1109 1.20E−152 coding region BB0027 [Borreliaf943.aa gi|2687905 (AE001116) B. burgdorferi predicted 2001 5.00E−273coding region BB0024 [Borrelia f943.aa gi|411592 L-sorbosonedehydrogenase 175 2.30E−15 [unidentified] f943.aa gnl|PID|d1006418L-sorbosone dehydrogenase 173 4.40E−15 [Acetobacter liquefaciens]f952.aa gi|2687880 (AE001115) glpE protein (glpE) 628 2.90E−84 [Borreliaburgdorferi] f07A.aa R33279 43 kD endoflagellum sheath protein. 1206.10E−25 f142.aa R95044 Apoptosis participating protein. 103 4.70E−18f147.aa W18209 Staphylococcus aureus Coenzyme A 194 4.80E−91 disulphidereductase (CoADR). f147.aa W06425 Water-forming NADH oxidase. 3698.00E−86 f147.aa R32089 Benzene dioxygenase polypeptide V. 104 4.70E−11f147.aa R66733 Aromatic dihydrodiol/catechol 105 9.00E−11 deoxygenase#5. f152.aa R81549 High affinity potassium uptake 137 3.70E−18transporter HKT1. f157.aa W15192 Staphylococcus aureus cell surface 2393.40E−37 protein. f17-6.aa W30763 Mannose-1-phosphate transferase 1785.20E−16 protein MNN4. f17-6.aa W03627 Human follicle stimulatinghormone 145 1.30E−11 GPR N-terminal sequence. f17-6.aa W03626 Humanthyrotropin GPR N-terminal 144 1.90E−11 sequence. f17-6.aa W21591Antibiotic potentiating peptide #3. 141 5.10E−11 f196.aa W05196Helicobacter pylori 50 kDa protective 183 2.70E−18 antigen G3.8. f196.aaW20916 H. pylori inner membrane protein 180 3.60E−17 14gp12015orf12.f196.aa W20287 H. pylori inner membrane protein, 169 6.50E−1524132293.aa. f196.aa W20769 H. pylori inner membrane protein, 1691.40E−14 07ee20513orf28. f196.aa W20767 H. pylori cytoplasmic protein,140 6.10E−14 07ee20513orf1. f197.aa W20769 H. pylori inner membraneprotein, 190 2.30E−19 07ee20513orf28. f197.aa W20287 H. pylori innermembrane protein, 190 2.00E−18 24132293.aa. f197.aa W05196 Helicobacterpylori 50 kDa protective 179 4.00E−16 antigen G3.8. f197.aa W20916 H.pylori inner membrane protein 182 6.30E−16 14gp12015orf12. f197.aaW20767 H. pylori cytoplasmic protein, 150 1.10E−12 07ee20513orf1.f21-4.aa R69629 B. burgdorferi OspF operon. 321 7.00E−39 f21-4.aa R89476B. burgdorferi OspG lipoprotein. 107 6.10E−34 f24-1.aa W22676 Borreliavariable major protein (VMP)- 412 4.60E−72 like protein VIsE. f291.aaW20152 H. pylori transporter protein, 336 1.70E−41 1464715.aa. f291.aaW24682 Helicobacter pylori transporter protein 234 8.20E−27 4882763.aa.f291.aa W20528 H. pylori cell envelope transporter 234 8.20E−27 protein4882763.aa. f291.aa W20592 H. pylori transporter protein, 168 7.60E−1701ce11513orf21. f301.aa W20287 H. pylori inner membrane protein, 1581.60E−13 24132293.aa. f301.aa W20916 H. pylori inner membrane protein158 1.90E−13 14gp12015orf12. f301.aa W20769 H. pylori inner membraneprotein, 158 2.40E−13 07ee20513orf28. f301.aa W05196 Helicobacter pylori50 kDa protective 157 2.80E−13 antigen G3.8. f301.aa W20767 H. pyloricytoplasmic protein, 138 4.30E−11 07ee20513orf1. f320.aa R24300Glycopeptide resistance protein VanY 142 2.90E−14 from E. faecium.f328.aa R15642 CTP synthetase. 274 3.00E−50 f328.aa W20778 H. pyloricytoplasmic protein, 122 1.90E−34 07ge20415orf6. f352.aa W03626 Humanthyrotropin GPR N-terminal 153 4.70E−12 sequence. f352.aa W21591Antibiotic potentiating peptide #3. 152 6.60E−12 f352.aa W03627 Humanfollicle stimulating hormone 145 5.30E−11 GPR N-terminal sequence.f4-50.aa W07187 B. garinii IP90 decorin binding protein. 305 1.30E−41f4-50.aa W07186 B. afzelii strain pGau decorin binding 161 1.60E−34protein. f4-50.aa W07185 B. burgdorferi HB-19 decorin binding 1732.80E−34 protein. f4-50.aa W07183 B. burgdorferi B31 decorin binding 1761.80E−33 protein. f4-50.aa W07190 B. burgdorferi JD1 decorin binding 1771.80E−33 protein. f4-50.aa W07182 B. burgdorferi 297 decorin binding 1771.10E−32 protein. f4-50.aa W07189 B. burgdorferi LP7 decorin binding 1771.10E−32 protein. f4-50.aa W07188 B. burgdorferi LP4 decorin binding 1773.90E−32 protein. f4-50.aa W07184 B. burgdorferi Sh.2.82 decorin binding177 1.30E−31 protein. f45-2.aa R89476 B. burgdorferi OspG lipoprotein.213 1.30E−35 f45-2.aa R70491 Leucocytozoan protozoa structural 2062.10E−20 protein epitope. f45-2.aa W03626 Human thyrotropin GPRN-terminal 211 6.10E−20 sequence. f45-2.aa W03627 Human folliclestimulating hormone 202 8.90E−19 GPR N-terminal sequence. f45-2.aaR69629 B. burgdorferi OspF operon. 111 1.10E−14 f45-2.aa W30763Mannose-1-phosphate transferase 166 1.00E−13 protein MNN4. f45-2.aaR97866 Chicken leucocytozoan immunogenic 154 7.10E−12 protein for use invaccines. f488.aa W15078 M. leprae gyrA precursor. 390 2.70E−143 f488.aaR88733 S. aureus mutant grlA protein. 698 6.70E−122 f488.aa R88731 S.aureus topoisomerase IV grlA 698 6.70E−122 subunit. f49-2.aa W22676Borrelia variable major protein (VMP)- 497 2.70E−75 like protein VlsE.f5-14.aa W03626 Human thyrotropin GPR N-terminal 234 6.60E−23 sequence.f5-14.aa W03627 Human follicle stimulating hormone 231 1.40E−22 GPRN-terminal sequence. f5-14.aa R70491 Leucocytozoan protozoa structural221 1.00E−20 protein epitope. f5-14.aa W30763 Mannose-1-phosphatetransferase 203 1.60E−18 protein MNN4. f5-14.aa R97866 Chickenleucocytozoan immunogenic 187 2.10E−15 protein for use in vaccines.f5-14.aa W21591 Antibiotic potentiating peptide #3. 176 4.60E−15f5-14.aa R69629 B. burgdorferi OspF operon. 106 3.50E−13 f5-14.aa R89476B. burgdorferi OspG lipoprotein. 157 6.20E−13 f5-14.aa W26536Trypanosoma cruzi antigen. 143 5.00E−11 f5-15.aa R69629 B. burgdorferiOspF operon. 448 1.30E−68 f5-15.aa R89476 B. burgdorferi OspGlipoprotein. 105 5.80E−24 f502.aa R69852 Ethylene response (ETR) mutant191 1.90E−35 protein etr1-3. f502.aa R69849 Ethylene response (ETR) geneproduct. 191 2.70E−35 f502.aa R69853 Ethylene response (ETR) mutant 1912.70E−35 protein etr1-4. f502.aa R69850 Ethylene response (ETR) mutant191 3.60E−35 protein etr1-1. f502.aa R69851 Ethylene response (ETR)mutant 191 3.60E−35 protein etr1-2. f502.aa R74632 QETR ethyleneresponse (ETR) protein 190 5.20E−26 from Arabidopsis thaliana. f502.aaR74629 Tomato ethylene response (TETR) 171 6.50E−23 protein. f502.aaR74633 Nr (never ripe) tomato ethylene 171 6.50E−23 response (ETR)protein. f502.aa R74630 Tomato TGETR1 ethylene response 123 1.20E−19protein. f51-2.aa W03626 Human thyrotropin GPR N-terminal 235 2.90E−23sequence. f51-2.aa R89476 B. burgdorferi OspG lipoprotein. 109 6.90E−23f51-2.aa W03627 Human follicle stimulating hormone 228 2.20E−22 GPRN-terminal sequence. f51-2.aa W30763 Mannose-1-phosphate transferase 2031.00E−18 protein MNN4. f51-2.aa R70491 Leucocytozoan protozoa structural191 7.50E−18 protein epitope. f51-2.aa R97866 Chicken leucocytozoanimmunogenic 183 4.80E−16 protein for use in vaccines. f51-2.aa W21591Antibiotic potentiating peptide #3. 159 6.20E−13 f51-2.aa R68838Plasmodium falciparum ABRA gene 142 1.10E−12 protein. f51-2.aa R27530Plasmodium falciparum blood and liver 142 2.80E−12 stage ABRA antigen.f51-2.aa W31186 Human p160 polypeptide 160.2. 148 2.30E−11 f51-2.aaW31185 Human p160 polypeptide 160.1. 148 2.40E−11 f517.aa W24296Staphylococcus aureus Gene #1 237 6.80E−30 polypeptide sequence 2.f541.aa R31013 P39-alpha. 1253 3.80E−229 f541.aa R33280 P39-beta. 5041.90E−117 f542.aa R33280 P39-beta. 711 3.20E−96 f542.aa R31013P39-alpha. 101 7.90E−16 f561.aa R69631 B. burgdorferi T5 protein. 9826.90E−131 f598.aa W20289 H. pylori transporter protein, 264 9.90E−3324218968.aa. f598.aa W20640 H. pylori transporter protein, 264 1.00E−3002ce11022orf8. f598.aa W20101 H. pylori transporter protein 233 8.50E−2711132778.aa. f598.aa W20861 H. pylori cell envelope transporter 2339.60E−27 protein, 12ge10305orf16. f598.aa W34202 Streptomyces effluxpump protein 196 2.80E−21 (frenolicin gene D product). f598.aa R71091 C.jejuni PEB1A antigen from ORF3. 168 1.20E−17 f600.aa W25527Staphylococcus aureus Gene #20 209 3.40E−26 polypeptide sequence 2.f600.aa W34201 Streptomyces efflux pump protein 169 6.50E−19 (frenolicingene C product). f600.aa W20639 H. pylori transporter protein, 1271.10E−14 02ce11022orf7. f603.aa W34200 Streptomyces efflux pump protein155 7.40E−32 (frenolicin gene B product). f604.aa R48035 Hyaluronic acidsynthase of 110 2.30E−20 Streptococcus equisimilis. f606.aa R48035Hyaluronic acid synthase of 116 1.20E−25 Streptococcus equisimilis.f607.aa R48035 Hyaluronic acid synthase of 141 1.50E−26 Streptococcusequisimilis. f631.aa W15192 Staphylococcus aureus cell surface 1607.30E−29 protein. f664.aa W20105 H. pylori flagella-associated protein,202 3.20E−46 1171928.aa. f664.aa W20688 H. pylori flagella-associatedprotein 202 2.60E−45 04ge11713orf5. f664.aa R97245 Virulence genecluster polypeptide 158 3.90E−13 product. f704.aa R60153Nematode-inducible transmembrane 104 2.50E−18 pore protein. f704.aaR33913 Sequence encoded by TobRB7-5A 104 2.50E−18 which encodes amembrane channel f704.aa R77082 Tobacco root specific promoter RB7 1042.50E−18 from clone lambda5A (TobRB7-5A). f742.aa W46499 Amino acidsequence of the spindly 101 2.50E−14 (SPY) protein of Arabidopsis.f752.aa W20733 H. pylori cell envelope protein, 141 3.00E−3706cp11722orf15. f752.aa W20358 H. pylori cell envelope protein 1104.20E−18 26366312.aa. f814.aa W20753 H. pylori transporter protein, 1787.90E−35 06gp11202orf7. f814.aa W20420 H. pylori cell envelopetransporter 160 2.30E−21 protein 33399142.aa. f843.aa R14319 HumanT-cell immunosuppressive 167 1.20E−19 factor. f860.aa W21894Asparaginyl-tRNA synthetase from 245 2.30E−38 Staphylococcus aureus.f860.aa W33903 Streptococcus pneumoniae asparaginyl 177 1.10E−22 tRNAsynthetase. f867.aa W34261 An alpha subunit of a thermostable 5921.30E−161 ATPase. f867.aa R10098 Alpha subunit of ATP-synthase. 7414.90E−144 f867.aa R31522 Carrot reverse transcriptase. 311 4.60E−130f867.aa R10099 Beta subunit of ATP-synthase. 121 7.90E−14 f867.aa W34262A beta subunit of a thermostable 116 1.00E−12 ATPase. f868.aa W34262 Abeta subunit of a thermostable 151 6.10E−109 ATPase. f868.aa R10099 Betasubunit of ATP-synthase. 172 1.90E−106 f868.aa W34261 An alpha subunitof a thermostable 117 3.10E−19 ATPase. f868.aa R10098 Alpha subunit ofATP-synthase. 113 2.00E−18 f868.aa R31522 Carrot reverse transcriptase.101 7.10E−15 f874.aa R10591 L-lactic acid dehyrogenase. 538 7.20E−109f874.aa R08355 Recombinant thermophilic NAD- 455 9.80E−99 dependantdehydrogenase. f874.aa R09295 Recombinant thermophilic NAD- 455 9.80E−99dependant dehydrogenase. f874.aa R15736 L-lactic acid dehydrogenase. 4261.60E−85 f874.aa P91948 Pig H4 isoenzyme. 393 2.00E−82 f874.aa W33108Chicken lactic acid dehydrogenase type 390 2.20E−80 B subunit. f874.aaW33107 Chicken lactic acid dehydrogenase type 385 1.10E−79 B subunit.f874.aa P80891 Testis-specific lactate dehydrogenase 339 5.50E−74subunit LDH-C4. f874.aa R94013 Heat resistant maleate dehydrogenase. 2551.30E−55 f874.aa R11119 Recombinant L-2-hydroxyisocaproic 224 7.90E−49acid dehydrogenase. f874.aa R62605 P. falciparum lactate dehydrogenase.255 2.00E−44 f874.aa W11476 Eimeria lactate dehydrogenase. 203 1.10E−25f943.aa P91223 Coenzyme-independent L-sorbosone 175 4.30E−16dehydrogenase from Gluconobacter

TABLE 3 Conservative Amino Acid Substitutions. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

TABLE 4 Residues Comprising Epito-Bearing Fragments Query ResiduesComprising Epito-Bearing Fragments f101.aa from about Lys-62 to aboutGly-64, from about Ser-111 to about Asp-113, from about Arg-136 to aboutArg-139, from about Pro-189 to about Asn-193. f11.aa from about Pro-38to about Lys-40, from about Glu-92 to about Lys-96. f12.aa from aboutPro-288 to about Asp-290, from about Asn-336 to about Gly-338, fromabout Tyr-410 to about Gly-413, from about Asp-418 to about Arg-420,from about Pro-552 to about Val-555, from about Gln-643 to aboutAsp-645, from about Gln-1061 to about Arg-1063, from about Asn-1130 toabout Lys-1132. f129.aa from about Glu-76 to about Arg-81, from aboutLys-144 to about Asn-146. f147.aa from about Gln-94 to about Thr-96.f152.aa from about Gly-35 to about Gly-37, from about Gln-321 to aboutGly-323. f154.aa from about Asn-39 to about Lys-41, from about Ser-74 toabout Lys-77, from about Ser-213 to about Gly-215, from about Ser-303 toabout Asp-306, from about Asp-422 to about Asn-424. f157.aa from aboutLys-21 to about Asp-24, from about Ser-45 to about Tyr-47. f17.aa fromabout Arg-17 to about Asn-20, from about Thr-94 to about Gly-96. f186.aafrom about Lys-305 to about Tyr-308. f196.aa from about Lys-121 to aboutAsn-123, from about Pro-278 to about Lys-282, from about Glu-576 toabout Tyr-578. f899.aa from about Asn-174 to about Asp-177. f925.aa fromabout Lys-201 to about Asp-204, from about Phe-291 to about Lys-294.f929.aa from about Pro-139 to about Asn-141, from about Arg-211 to aboutGlu-214, from about Thr-370 to about Asn-375. f933.aa from about Ser-139to about Lys-143. f940.aa from about Gly-143 to about Asn-148. f943.aafrom about Asp-58 to about Asp-60, from about Lys-157 to about Asn-159,from about Asp-217 to about Asp-221, from about Lys-250 to aboutAsn-254, from about Pro-262 to about Asn-264, from about Gly-305 toabout Trp-307. f952.aa from about Ser-52 to about Ser-54. f4.aa fromabout Arg-64 to about Arg-67. f43.aa from about Ser-84 to about Gln-87,from about Asp-231 to about Tyr-233, from about Arg-296 to aboutAsp-300. f50.aa from about Glu-136 to about Gly-138, from about Asp-153to about Lys-155, from about Asp-289 to about Asp-291, from aboutGlu-458 to about Asn-461. f65.aa from about Glu-120 to about Asp-122,from about Pro-204 to about Tyr-206. f8.aa from about Pro-263 to aboutArg-265, from about Asp-274 to about Lys-278. f82.aa from about Tyr-66to about Gly-68, from about Ser-116 to about Lys-119, from about Asp-121to about Gly-123, from about Pro-128 to about Gly-131. f86.aa from aboutAsn-179 to about Asn-181, from about Lys-192 to about Asn-194, fromabout Lys-270 to about Asn-272, from about Lys-279 to about Lys-282,from about Asp-331 to about Asn-333. f477.aa from about Pro-250 to aboutLys-253. f488.aa from about Lys-76 to about Lys-79, from about Asn-486to about Asp-489, from about Lys-508 to about Gly-510, from aboutAsn-559 to about Gly-562. f494.aa from about Lys-76 to about Asn-78.f516.aa from about Lys-32 to about Asp-34. f523.aa from about Pro-202 toabout Asn-206, from about Lys-255 to about Tyr-258. f526.aa from aboutAsn-85 to about Lys-88, from about Asp-136 to about Gly-138. f577.aafrom about Cys-18 to about Lys-22, from about Asn-297 to about Gln-300.f584.aa from about Pro-131 to about Lys-133, from about Pro-200 to aboutSer-202. f596.aa from about Arg-42 to about Asp-44, from about Asp-117to about Tyr-119, from about Pro-205 to about Asp-207. f600.aa fromabout Pro-143 to about Asp-145. f603.aa from about Phe-35 to aboutSer-37. f607.aa from about Gln-67 to about Lys-70, from about Asp-273 toabout Tyr-275, from about Asp-333 to about Gly-338, from about Pro-359to about Lys-362, from about Arg-409 to about Gly-411. f611.aa fromabout Arg-133 to about Gly-135. f631.aa from about Pro-132 to aboutAsn-136, from about Asn-159 to about Tyr-161, from about Pro-216 toabout Asp-218, from about Pro-220 to about Lys-223. f688.aa from aboutLys-266 to about Asp-268, from about Lys-271 to about Asn-273, fromabout Lys-315 to about Lys-318. f704.aa from about Lys-250 to aboutLys-253. f707.aa from about Lys-131 to about Asp-134, from about Asp-246to about Asn-249. f709.aa from about Tyr-39 to about Gly-42, from aboutLys-148 to about Gly-150, from about Arg-269 to about Gly-272, fromabout Ser-466 to about Tyr-468, from about Asn-489 to about Asn-491,from about Lys-575 to about Asp-578, from about Pro-642 to aboutLys-644. f197.aa from about Pro-217 to about Asp-219, from about Glu-675to about Asp-678, from about Pro-687 to about Asn-689, from aboutGlu-694 to about Gln-696. f200.aa from about Arg-174 to about Phe-179.f208.aa from about Arg-326 to about Ser-328. f210.aa from about Pro-191to about Ile-194. f221.aa from about Asn-133 to about Asn-135. f253.aafrom about Arg-191 to about Gly-194. f269.aa from about Ser-271 to aboutThr-273, from about Asp-284 to about Gly-286. f29.aa from about Pro-159to about Ser-161. f290.aa from about Pro-240 to about Gly-244. f291.aafrom about Gln-267 to about Lys-269. f296.aa from about Glu-98 to aboutLys-101. f3.aa from about Asn-241 to about Lys-245. f30.aa from aboutAsn-156 to about Tyr-159, from about Asn-178 to about Lys-180. f939.aafrom about Ser-245 to about Asn-249. f739.aa from about Asn-80 to aboutTyr-82, from about Lys-208 to about Ser-210. f742.aa from about Ser-141to about Asp-145, from about Asn-222 to about Gln-225, from aboutAsp-243 to about Tyr-247, from about Asn-249 to about Asn-251. f743.aafrom about Arg-111 to about Gly-114, from about Pro-131 to aboutAsp-134. f790.aa from about Thr-40 to about Asn-42, from about Ser-53 toabout Ser-55, from about Lys-215 to about Asp-218, from about Asn-274 toabout Gly-277. f792.aa from about Val-82 to about Ser-84, from aboutSer-102 to about Asn-104, from about Gln-127 to about Tyr-130, fromabout Lys-309 to about Asn-314, from about Lys-375 to about Thr-377,from about Pro-511 to about His-513, from about Thr-515 to aboutAsp-517. f797.aa from about Pro-119 to about Gly-122, from about Lys-166to about Asn-169. f799.aa from about Asn-31 to about Asn-34, from aboutGln-44 to about Asn-47, from about Pro-123 to about Gly-125. f814.aafrom about Ser-120 to about Ser-122, from about Arg-636 to aboutAsn-638, from about Cys-967 to about Ser-969. f820.aa from about Thr-563to about Tyr-565. f850.aa from about Tyr-159 to about Tyr-164, fromabout Gln-375 to about Asp-379. f853.aa from about Thr-180 to aboutLys-184, from about Arg-231 to about Asp-233, from about Asn-252 toabout Gly-254. f859.aa from about Lys-46 to about Ser-52, from aboutPro-88 to about Asn-91, from about Asn-117 to about Asp-120. f861.aafrom about Asp-38 to about Lys-40, from about Lys-219 to about Asn-225.f368.aa from about Gln-228 to about Asn-231. f371.aa from about Tyr-109to about Asn-111, from about Asn-162 to about Gln-164. f502.aa fromabout Asn-118 to about Lys-122, from about Ser-269 to about Gly-271,from about Lys-370 to about Asp-373, from about Asn-509 to aboutLys-511, from about Lys-705 to about Arg-707, from about Thr-912 toabout Gly-914, from about Pro-1213 to about Asp-1216, from aboutAsn-1491 to about Arg-1493. f527.aa from about Cys-20 to about Gln-22,from about Asn-38 to about Asn-40, from about Phe-112 to about Asp-114,from about Lys-160 to about Asn-162, from about Ser-199 to aboutAsp-201, from about Gln-258 to about Gly-261, from about Arg-282 toabout Asn-284, from about Ser-297 to about Asp-299. f541.aa from aboutSer-68 to about Asn-71. f604.aa from about Lys-77 to about Gly-79, fromabout Lys-201 to about Asn-203, from about Asp-252 to about Asp-254,from about Tyr-347 to about Gly-350, from about Asp-514 to aboutTrp-516. f736.aa from about Lys-20 to about Asn-24, from about Arg-147to about Ser-153, from about Ser-231 to about Lys-233. f752.aa fromabout Thr-119 to about Lys-122, from about Pro-420 to about Gly-422.f798.aa from about Asp-33 to about Thr-36, from about Lys-180 to aboutHis-183. f635.aa from about Pro-100 to about Asn-102, from about Asp-145to about Phe-147. f32.aa from about Lys-18 to about Asn-20. f320.aa fromabout Asn-193 to about Leu-195, from about Gln-248 to about Lys-250.f352.aa from about Ser-46 to about Asn-49. f301.aa from about Lys-178 toabout Lys-180, from about Ser-401 to about Tyr-404. f373.aa from aboutGly-88 to about Lys-90, from about Asn-539 to about Lys-542, from aboutGlu-654 to about Ser-657. f384.aa from about Pro-250 to about Asn-252,from about Asp-266 to about Lys-268. f446.aa from about Asp-20 to aboutSer-26, from about Asn-146 to about Lys-149. f542.aa from about Arg-86to about Gly-88, from about Arg-163 to about Asn-165. f93.aa from aboutAsn-152 to about Asp-155. f105.aa from about Asp-48 to about Phe-50.f150.aa from about Thr-214 to about Asp-218, from about Asp-256 to aboutAsp-259. f219.aa from about Asn-77 to about Asn-81, from about Asp-111to about Asn-115. f229.aa from about Gln-61 to about Asn-63. f32.aa fromabout Lys-18 to about Asn-20. f186.aa from about Lys-305 to aboutTyr-308. f216.aa from about Ser-105 to about Asn-107. f328.aa from aboutAsn-105 to about Asp-107. f352.aa from about Ser-46 to about Asn-49.f867.aa from about Thr-3 to about Gly-5, from about Lys-156 to aboutSer-159. f868.aa from about Arg-94 to about Gly-96, from about Pro-257to about Gly-261, from about Pro-295 to about Asp-297, from aboutArg-340 to about Asp-342. f872.aa from about Ser-19 to about Lys-23,from about Thr-139 to about Asp-142, from about Ser-282 to aboutTyr-286, from about Ser-311 to about Ser-313. f886.aa from about Thr-83to about Asp-85, from about Asp-106 to about Lys-108, from about Lys-143to about Gly-147, from about Asp-186 to about Asn-191. f888.aa fromabout Asn-65 to about Asp-67. f893.aa from about Asn-203 to aboutAsn-207, from about Thr-446 to about Asn-450. f605.aa from about Arg-31to about Asp-33. f606.aa from about Asn-68 to about Gly-71, from aboutAsn-136 to about Lys-139, from about Asn-223 to about Tyr-226, fromabout Ser-276 to about Tyr-279, from about Pro-362 to about Asn-365,from about Arg-503 to about Trp-507. f679.aa from about Lys-154 to aboutAsp-156, from about Lys-224 to about Arg-226, from about Asn-260 toabout Asp-264, from about Glu-363 to about Lys-366, from about Asp-387to about Gly-389, from about Tyr-441 to about Lys-443, from aboutArg-501 to about Tyr-504. f11-12.aa from about Pro-91 to about Asn-93,from about Pro-181 to about Asp-186, from about Lys-244 to aboutSer-248. f11-4.aa from about Asn-160 to about Lys-163. f14-8.aa fromabout Pro-92 to about Gln-95, from about Lys-123 to about Thr-125, fromabout Lys-215 to about Asp-219. f17-6.aa from about Pro-36 to aboutGlu-38. f19-2.aa from about Ser-104 to about Ser-106, from about Gln-230to about Asn-232. f19-4.aa from about Val-79 to about Thr-82, from aboutPro-195 to about Gly-201. f19-6.aa from about Asp-24 to about Lys-30,from about Pro-36 to about Glu-38. f21-4.aa from about Cys-24 to aboutAsn-26. f28-2.aa from about Ser-77 to about Lys-80, from about Tyr-274to about Asn-277. f28-3.aa from about Glu-53 to about Arg-57, from aboutGln-82 to about Asn-85, from about Gln-157 to about Asn-159. f31-2.aafrom about Arg-95 to about Arg-97, from about Asn-297 to about Asn-299.f4-15.aa from about Pro-182 to about Asp-184, from about Lys-220 toabout Asp-222. f4-50.aa from about Thr-109 to about Asn-111. f42-1.aafrom about Asn-55 to about Asn-57, from about Arg-81 to about Ser-84,from about Asp-94 to about Asn-97. f45-2.aa from about Asn-83 to aboutGly-86. f47-2.aa from about Ser-29 to about Asp-33, from about Asn-94 toabout Lys-99, from about Pro-152 to about Lys-157. f49-2.aa from aboutAsn-452 to about Gly-454. f5-14.aa from about Glu-102 to about Asp-106,from about Thr-272 to about Asn-275, from about Glu-313 to aboutAsn-315, from about Ser-370 to about Ser-372. f5-15.aa from aboutLys-170 to about Gly-173, from about Asn-194 to about Gly-196. f51-2.aafrom about Asp-302 to about Lys-304. f6-21.aa from about Glu-38 to aboutAsn-42, from about Arg-84 to about Gly-87. f6-27.aa from about Asp-67 toabout Asn-69, from about Arg-85 to about Asn-89, from about Lys-168 toabout Gly-171, from about Lys-179 to about Asn-181, from about Ser-380to about His-382. f6-5.aa from about Ser-67 to about Asn-71. f7-30.aafrom about Pro-94 to about Asp-96, from about Lys-144 to about Arg-147.f76-1.aa from about Asn-30 to about Lys-35, from about Lys-113 to aboutGly-116, from about Glu-119 to about Lys-121. f8-10.aa from about Pro-25to about Lys-32, from about Ser-168 to about Thr-172. f01a.aa_bb001 fromabout Pro-123 to about Asp-125, from about Ser-179 to about Asp-181,from about Lys-255 to about Gly-259. _bb0011 from about Ala8 about Arg17, from about Tyr31 to about Gly40, from about Ser65 to about Lys78,from about Val93 to about Asp102, from about Ser120 to about Ile129,from about Pro156 to about Glu170, from about Lys187 to about Asn 196,from about His205 to about Lys214, from about Gly226 to about Glu235,fro about Gln253 to about Asn266, from about Glu283 to about Glu293,from about Leu311 to about Ile320, from about Arg326 to about Gly335,from about Pro340 to about Ala349 f02a.aa_bb002 from about Tyr-169 toabout Asn-171, from about Tyr-242 to about Asn-245, from about Lys-264to about Asp-267. _bb9 from about Met7 to about Lys16, from about Lys47to about Ser57, from about Asn80 to about Ser89, from about Gly103 toabout Glu113, from about Lys125 to about Pro133, from about Lys138 toabout Ala147 f03a.aa_bb006 from about Asp-54 to about Thr-57, from aboutLys-201 to about His-204. _bb014 from about Pro23 to about Gln31, fromabout Ser37 to about Asp45, from about Leu76 to about Asn84, from aboutLeu76 to about Val84, from about Ser89 to about Asn97, from about Ser105to about Lys113, from about Asn120 to about Met128, from about Asn159 toabout Gly 167, from about Lys173 to about Bal181 _bb023 from about Asp17to about Gly27, from about Arg40 to about Asp48, from about Val64 toabout Asp72, from about Glu105 to about Thr113, from about Ser141 toabout Gly150, from about Asp155 to about Ile163, from about Asn184 toabout Lys198, from about Ile219 to about Pro227, from about Ser230 toabout Phe238, from about Ser241 to about Asn250, from about Asp270 toabout Val278, from about Ser285 to about Leu293, from about Glyu307 toabout Ser315, from about Lys327 to about Asn335 f08a.aa_bb024 from aboutAsn-30 to about Asp-33, from about Ser-116 to about Asn-118, from aboutAsn-154 to about Gly-156. f09a.aa_bb025 from about Asn-30 to aboutSer-35, from about Thr-145 to about Asn-148.

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence selected from the group consisting of: (a) anucleotide sequence encoding any one of the amino acid sequences of thepolypeptides shown in Table 1; (b) a nucleotide sequence complementaryto any one of the nucleotide sequences in (a); (c) a nucleotide sequenceat least 95% identical to any one of the nucleotide sequences shown inTable 1; and (d) a nucleotide sequence at least 95% identical to anucleotide sequence complementary to any one of the nucleotide sequencesshown in Table
 1. 2. An isolated nucleic acid molecule of claim 1comprising a polynucleotide which hybridizes under stringenthybridization conditions to a polynucleotide having a nucleotidesequence identical to a nucleotide sequence in (a) or (b) of claim
 1. 3.An isolated nucleic acid molecule of claim 1 comprising a polynucleotidewhich encodes an epitope-bearing portion of a polypeptide in (a) ofclaim
 1. 4. The isolated nucleic acid molecule of claim 3, wherein saidepitope-bearing portion of a polypeptide comprises an amino acidsequence listed in Table
 4. 5. A method for making a recombinant vectorcomprising inserting an isolated nucleic acid molecule of claim 1 into avector.
 6. A recombinant vector produced by the method of claim
 5. 7. Ahost cell comprising the vector of claim
 6. 8. A method of producing apolypeptide comprising: (a) growing the host cell of claim 7 such thatthe protein is expressed by the cell; and (b) recovering the expressedpolypeptide.
 9. An isolated polypeptide comprising a polypeptideselected from the group consisting of: (a) a polypeptide consisting ofone of the complete amino acid sequences of Table 1; (b) a polypeptideconsisting of one the complete amino acid sequences of Table 1 exceptthe N-terminal residue; (c) a fragment of the polypeptide of (a) havingbiological activity; and (d) a fragment of the polypeptide of (a) whichbinds to an antibody specific for the polypeptide of (a).
 10. Anisolated antibody specific for the polypeptide of claim
 9. 11. Apolypeptide produced according to the method of claim
 8. 12. An isolatedpolypeptide comprising an amino acid sequence at least 95% identical tothe polypeptide of claim
 9. 13. An isolated polypeptide antigencomprising an amino acid sequence of an B. burgdorferi epitope shown inTable
 4. 14. An isolated nucleic acid molecule comprising apolynucleotide with a nucleotide sequence encoding a polypeptide ofclaim
 9. 15. A hybridoma which produces an antibody of claim
 10. 16. Avaccine, comprising: (1) one or more B. burgdorferi polypeptidesselected from the group consisting of a polypeptide of claim 9; and (2)a pharmaceutically acceptable diluent, carrier, or excipient; whereinsaid polypeptide is present, in an amount effective to elicit protectiveantibodies in an animal to a member of the Borrelia genus.
 17. A methodof preventing or attenuating an infection caused by a member of theBorrelia genus in an animal, comprising administering to said animal apolypeptide of claim 9, wherein said polypeptide is administered in anamount effective to prevent or attenuate said infection.
 18. A method ofdetecting Borrelia nucleic acids in a biological sample obtained from ananimal, comprising a process selected from the group consisting of: (a)contacting the sample with one or more nucleic acids of claim 1, underconditions such that hybridization occurs, and detecting hybridizationof said nucleic acids to the one or more Borrelia nucleic acid sequencespresent in the biological sample; and (b) amplifying one or moreBorrelia nucleic acid sequences in said sample using polymerase chainreaction, and detecting said amplified Borrelia nucleic acid.
 19. A kitfor detecting Borrelia antibodies in a biological sample obtained froman animal, comprising (a) a polypeptide of claim 9 attached to a solidsupport; and (b) detecting means.
 20. A method of detecting Borreliaantibodies in a biological sample obtained from an animal, comprising(a) contacting the sample with a polypeptide of claim 9; and (b)detecting antibody-antigen complexes.